| Chitin and its derivatives have great research and application value,because of its good biocompatibility,biodegradability,low toxicity and various biological activities.In recent years,thermosensitive chitin derivatives,such as thermosensitive carboxymethyl chitin(CMCH)and hydroxypropyl chitin(HPCH)have received extensive attention.They can be used as injectable hydrogels in biomedical fields such as minimally invasive in situ drug delivery,bio-ink for 3D printing,high-end implanted medical devices and tissue engineering in situ repair.However,a small amount of heterologous protein remaining in chitin and its derivatives may cause serious immune reactions in the human body.Therefore,regulatory agencies had set a criterion that the content of protein in medical chitin derivative materials should be not more than 0.2%.However,there are not many research reports about protein determination of chitin derivatives,of which the protein assays are mainly based on colorimetry.In this work,the protein content of carboxymethyl chitosan(CMCS),CMCH and HPCH for biomedical applications was quantitatively determined.The main research contents of this thesis are listed as follows.(1)Concerning the precipitation issue of the mixing Bradford reagent with CMCS or CMCH solution,we put forward some improvement measures to avoid precipitation and maintain high test sensitivity through optimization of dye reagent composition and test conditions,such as the ratio of phosphoric/hydrochloric acid,the amount of ethanol and the incubation time.The modified Bradford method has a good linear relationship within protein concentration of 5-40 mg/L.The recovery is between 95% and 114%,and the relative standard deviation is lower than 6%.Finally,we discussed a possible interference,which may be caused by the interaction between CMCH and dye molecules.It provides a certain basis of experimental and theoretical for subsequent research,but more experimental data are still needed for systematic analysis and verification.(2)In order to establish a simple and reliable method to determine the residual protein content in HPCH samples,We explored the three potential methods(Bradford,Lowry and BCA methods).It was found that both Bradford and Lowry methods are not suitable for the protein assay in the HPCH sample,because the protein concentration in HPCH solution by the Bradford assay is not directly proportional to the polymer concentration and precipitation occurred in the HPCH solution incubated with Folin-phenol reagent.In the protein analysis of HPCH by the BCA method,the BCA method is suitable for a lower concentration of thermosensitive HPCH solution(1-2 mg/m L),while a higher concentration of thermosensitive HPCH solution will result in micellization under the test conditions and interfere with the protein assay.We had proposed a modified BCA method through the first order derivative of the absorption value to eliminate the interference of light scattering.The modified BCA method has a good linear relationship within the protein concentration of 2-14 mg/L.The recovery is between 95% and 105%,and the relative standard deviation is lower than 5%.The limit of detection and the limit of quantitation were 0.91 mg/L and 2.75mg/L,respectively.It fully meets the criteria of the Chinese Pharmacopoeia drug quality standard analytical method validation guidelines. |
