石墨相氮化碳特异性吸附血液中血红蛋白的条件优化

Optimization of specific adsorption conditions for hemoglobin in blood by graphite-phase carbon nitride

背景:心脏外科体外循环的安全时限仅6 h的主要原因是游离血红蛋白堆积,目前临床尚无有效的解决办法。目的:利用石墨相氮化碳可特异性吸附游离血红蛋白的特性,探究其可特异性吸附血红蛋白的最佳条件可达到的最高吸附效率。方法:配制不同质量浓度的血红蛋白溶液,研究石墨相氮化碳对血红蛋白的吸附能力。基于这些结果,考虑临床应用条件,探究pH值、石墨相氮化碳∶血样、反应温度、反应时间这4个单因素对吸附效率的影响。应用Design-Expert.8.0.6响应面分析软件提供的模型,设pH值、石墨相氮化碳∶血样、反应温度、反应时间4个因素为自变量,全血中游离血红蛋白吸附效率为因变量,设计四因素三水平实验,根据单因素实验结果选定三因素的零水平和波动区,检测吸光度值,计算血红蛋白吸附效率。利用电泳验证石墨相氮化碳对全血中血红蛋白的特异性吸附效果。结果与结论:①随着血液中血红蛋白质量浓度的增大,石墨相氮化碳对游离血红蛋白的吸附量也逐渐增大,但不满足线性关系;②单因素实验显示,pH=8.0时,石墨相氮化碳对游离血红蛋白的吸附效率为93.5%,对其他蛋白吸附很少,当pH值进一步升高时,对游离血红蛋白的吸附效率先维持平稳后逐渐下降;随着石墨相氮化碳∶血样比值、反应温度及反应时间的增加,石墨相氮化碳对游离血红蛋白的吸附效率呈现上升趋势;③通过Design-Expert.8.0.6软件计算实验的多元回归方程,可以得到4个因素的最佳作用值:pH=7.5,石墨相氮化碳∶血样为6.00 mg∶1 mL,反应温度为36.5℃,反应时间为60 min,全血中游离血红蛋白的吸附效率为54.34%;SDS-聚丙烯酰胺凝胶实验,在最佳吸附条件下,石墨相氮化碳可实现对全血中血红蛋白的特异性吸附;④结果显示,石墨相氮化碳可特异性吸附游离血红蛋白,且在最佳吸附条件下可提高吸附效率,有望作为一种新型生物医用材料进一步推动组织工程材料的发展。

BACKGROUND:Currently,there has been no effective strategy for prolonging the 6-hour safe time limit of cardiopulmonary bypass mainly due to the accumulation of free hemoglobin.OBJECTIVE:To investigate the optimum conditions and the highest adsorption efficiency of graphite-phase carbon nitride(g-C3N4)adsorbing free hemoglobin.METHODS:Different concentrations of hemoglobin solutions were prepared to study the adsorption capacity of g-C3N4 for hemoglobin,and then the adsorption efficiency of characteristic proteins(albumin,hemoglobin,cytochrome C)in whole blood at different pH values was explored.Based on clinical application conditions,the influences of pH,g-C3N4:blood sample(mg/mL),reaction temperature(°C)and reaction time(minite)on adsorption efficiency were investigated.Using the model created by the response surface analysis software Design-Expert.8.0.6,pH(A),g-C3N4:blood sample(mg/mL)(B),reaction time(C),and reaction temperature(D)were set as independent variables,and the adsorption efficiency of free hemoglobin in whole blood as dependent variables,the experiment with four factors and three levels was designed.According to the single factor experiment results,the zero level and fluctuation zone of three factors were selected,and the absorbance was measured by ultraviolet spectrophotometer to calculate the content of free hemoglobin before and after adsorption,and then the adsorption efficiency was calculated.Finally,the specific adsorption effect of g-C3N4 on hemoglobin in whole blood was verified by electrophoresis.RESULTS AND CONCLUSION:(1)With the increase of hemoglobin concentration in blood,the adsorption capacity of g-C3N4 to free hemoglobin increased gradually,but did not satisfy the linear relationship.(2)Single factor experiment showed that when the pH was 8.0,the adsorption efficiency of free hemoglobin in whole blood was 93.5%,and the adsorption of other proteins was few.When the pH value increases further,the adsorption efficiency for free hemoglobin initially remains stable and then gradually decreases.With the increase of g-C3N4:blood sample ratio,reaction time and reaction temperature,the adsorption efficiency of free hemoglobin in whole blood increased gradually.(3)The multiple regression equation of this experiment was calculated by Design-Expert.8.0.6 software,and the optimal action values of the four factors were obtained.The highest adsorption efficiency of free hemoglobin in whole blood was obtained by software analysis under the following experimental conditions:pH=7.5,in g-C3N4:blood sample(mg/mL)was 6.00:1,reaction temperature was 36.5°C,reaction time was 60 minutes,and the adsorption efficiency of free hemoglobin in whole blood was 54.34%.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that g-C3N4 achieved specific adsorption of hemoglobin in whole blood under the optimum adsorption conditions.(4)These results indicate that g-C3N4 can specifically adsorb free hemoglobin and improve the adsorption efficiency under the optimal adsorption conditions,which is expected to be a new biomedical material and further promote the development of tissue engineering materials.