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固定化金属离子亲和层析(IMAC)在生物分离中得到广泛的应用,但介质存在着较强的非特异性吸附,导致亲和介质分离效率下降。为减少IMAC的非特异性吸附,选用聚乙二醇单甲醚5000(m PEG 5000)为原料,经醛基化后修饰磁性壳聚糖微球固定化Cu2+亲和介质(MCS@Cu2+),得到m PEG修饰固定化Cu2+亲和介质(m PEG@MCS@Cu2+)。以牛血清白蛋白(BSA)作为大分子模型,以修饰的亲和介质对BSA吸附阻拒率为指标,优化了m PEG修饰MCS@Cu2+的工艺条件。结果表明:在反应温度为40℃、反应时间为20 h、n-CHO:n-NH2为4:1时,在m PEG@MCS@Cu2+上修饰的m PEG含量最大为12.5%,与MCS@Cu2+相比,修饰介质对BSA吸附阻拒率可达到37.4%。说明经m PEG修饰的MCS@Cu2+亲和介质在应用于富集活性分子时,可有效减少大分子非特异性吸附,提高IMAC分离效率。
Immobilized metal ion affinity chromatography (IMAC) has been widely used in biological separation, but the medium has strong nonspecific adsorption, leading to the decline of the affinity medium separation efficiency. In order to reduce non-specific adsorption of IMAC, polyethylene glycol monomethyl ether 5000 (m PEG 5000) was used as the raw material to immobilize the magnetic chitosan microsphere to immobilize Cu2 + affinity medium (MCS @ Cu2 +). m PEG modified immobilized Cu2 + affinity medium (m PEG @ MCS @ Cu2 +). Using bovine serum albumin (BSA) as a macromolecule model, the adsorption affinity of BSA to the modified affinity media was used as an index to optimize the process conditions of m PEG modified MCS @ Cu2 +. The results showed that the m PEG content modified by m PEG @ MCS @ Cu2 + was up to 12.5% at the reaction temperature of 40 ℃, the reaction time of 20 h and the n-CHO: n-NH2 ratio of 4: Compared with Cu2 +, BSA adsorption inhibition rate of modified media can reach 37.4%. It indicates that mPEG modified MCS @ Cu2 + affinity medium can effectively reduce the nonspecific adsorption of macromolecules and improve the efficiency of IMAC separation when applied to enrichment of active molecules.