Separation and purification of target proteins are essential for drug discovery and development. A magnetic separation has been exploited due to its high efficiency. Despite many studies on chemical crosslinking of a magnetic agarose microsphere (MAM), the study on a crosslinking reaction preventing MAM from hydrolytic degradation during the reaction and the effects of the crosslinks on the physicochemical and electrochemical properties of the crosslinked MAM (CL-MAM) has rarely been reported. In our study, CL-MAM was prepared using a sequential crosslinking by which MAM was crosslinked using epichlorohydrin (ECH) in DMSO after crosslinking the MAM by 1,3-dichloro-2-propanol (DCP) for a short time. Such a crosslinking prevented the hydrolytic degradation of DCP-crosslinked MAM (D-CL-MAM) and DCP and ECH-crosslinked MAM (DE-CL-MAM). MAM had a mean diameter of 110 ± 57.42 µm and its morphology was not affected by the crosslinking. In FT-IR analysis, increase of peak intensity at 1037 cm⁻¹ confirmed newly formed C-O-C bonds in the agarose of CL-MAM. Thermal stability of MAM increased by both DCP crosslinking and subsequent ECH crosslinking of the agarose. While T_g of MAM was not shifted by the crosslinking, the onset temperatures of glass transition and thermal decomposition increased by DCP crosslinking and further increased by DCP and ECH-crosslinking. Stiffness (S) and elastic modulus (E) of MAM were enhanced by DCP crosslinking and subsequent ECH crosslinking, indicating a half of ECH formed crosslinks between agarose mainchains while the other half crosslinked adjacent pyranose rings in the agarose mainchain. Permittivity analysis revealed that α relaxation temperature of the agarose film corresponded to T_g determined by DSC analysis and the relaxation intensity decreased with increasing crosslink density of the agarose. The higher swelling ratio of DE-CL-MAM compared to D-CL-MAM was attributed to greater S and E values of DE-CL-MAM. The hydrolysis-repressive sequential crosslinking of MAM using DCP and ECH can be a feasible approach to modulate physicochemical and electrochemical properties of MAM.

目标蛋白的分离和纯化对于药物发现和开发至关重要。磁分离因其高效率而被开发出来。尽管对磁性琼脂糖微球 (MAM) 的化学交联进行了许多研究，但关于防止 MAM 在反应过程中水解降解的交联反应以及交联对交联 MAM (CL-MAM) 的物理化学和电化学性能的影响的研究很少被报道。在我们的研究中，CL-MAM 是使用顺序交联制备的，其中 MAM 在用 1,3-二氯-2-丙醇 (DCP) 短时间交联 MAM 后，在 DMSO 中使用表氯醇 (ECH) 进行交联。这种交联阻止了 DCP 交联 MAM 的水解降解（D-CL-MAM) 和 DCP 和 ECH 交联的 MAM (DE-CL-MAM)。MAM 的平均直径为 110 ± 57.42 µm，其形态不受交联的影响。在 FT-IR 分析中，1037 cm ^-1处峰强度的增加证实了 CL-MAM 琼脂糖中新形成的 COC 键。MAM 的热稳定性通过琼脂糖的 DCP 交联和随后的 ECH 交联而增加。虽然_TgMAM 没有因交联而改变，玻璃化转变和热分解的起始温度因 DCP 交联而增加，并因 DCP 和 ECH 交联而进一步增加。MAM 的刚度 (S) 和弹性模量 (E) 通过 DCP 交联和随后的 ECH 交联增强，表明一半 ECH 在琼脂糖主链之间形成交联，而另一半在琼脂糖主链中交联相邻的吡喃糖环。介电常数分析表明，琼脂糖膜的α弛豫温度对应于由DSC分析确定的_Tg，弛豫强度随着琼脂糖交联密度的增加而降低。与D相比，DE-CL-MAM 的溶胀率更高-CL-MAM 归因于 DE-CL-MAM 更大的 S 和 E 值。使用 DCP 和 ECH 对 MAM 进行水解抑制顺序交联可能是调节 MAM 物理化学和电化学性质的可行方法。