Editing Magnetic beads Preparation Methods (section)

== Agarose coated magnetic beads ==

称取4g琼脂糖粉，加入40mL水，加热溶解，得到10%的琼脂糖溶液，降温至65 °C左右，向其中缓慢加入40%Na0H溶液0.5mL和双功能交联剂烯丙基缩水甘油醚6mL。其中，烯丙基缩水甘油醚在水相中的浓度为15%，0H—的浓度为0.21mol/L。在65°C下搅拌反应8h后，用60%的冰醋酸溶液调节pH值7〜8终止反应，得到了烯丙基化的琼脂糖溶液。向溶液中加入4倍体积的乙醇，离心收集沉淀，_70°C下预冻2小时，冷冻干燥72小时得到修饰的琼脂糖原料。

[0070] (2)共混制备交联剂修饰的琼脂糖微球

[0071] 准确称取修饰的琼脂糖原料1.20g (占共混原料总质量的50%)，未修饰琼脂糖原料1.20g (占共混原料总质量的50%)，共同加热溶解于60mL去离子水中，配成4%的共混琼脂糖溶液作为分散相。向600mL液体石蜡/石油醚的混合溶液中(V液体石蜡:V石油醚=11:1)，加入6%的油溶性乳化剂Span80，作为连续相。将油相预热至60°C，在自身重力作用下，分散相缓慢通过平均孔径25.9 μ m的疏水性SPG微孔膜进入到连续相中，形成均一的W/0型乳液。整个乳化过程在60°C下完成，制备期间用磁力搅拌使乳液保持均匀分散。膜乳化过程结束后，将乳液转入三口瓶中，在90rpm的搅拌转速下，将所得W/0型乳液缓慢降温至20°C以下，使其固化形成微球，并依次用石油醚、乙醇及蒸馏水洗涤得到浓度为4%的修饰琼脂糖微球。

[0072] ( 3 )琼脂糖微球内部交联

[0073] 将20g修饰琼脂糖微球分散于40g水中,加入2gNaAc,搅拌5min,滴加溴水至溶液呈深黄色，持续Imin不变色，继续反应15min。然后，加入甲酸钠至凝胶呈白色，水洗三次。将该溴化凝胶重新分散于20mL水中，加入2gNa2S04，搅拌15min后，加入40%Na0H溶液2mL(含有5%NaBH4)，反应2h。然后转入恒温水浴振荡器中，37.5°C振荡反应12h后，水洗至中性，得到浓度为4%的高交联琼脂糖微球。采用上述共混法制备的琼脂糖微球球形圆整(扫描电子显微镜如图1所示)，平均粒径85.33 μ m。

取7.5g琼脂糖于250mL水中(琼脂糖水溶液浓度为3%)，常温搅拌lh，加入

0.12gKCl继续搅拌0.5h后，加热至琼脂糖沸腾，溶液呈现透明状。准备1.5L甲苯溶液，加入23.49g聚醋酸乙烯酯和2.349g油酸。将有机相于70°C下恒温搅拌至混合均匀，将沸腾的琼脂糖水溶液倒入有机相中，一定搅拌下琼脂糖的分散乳化lh。一次性加入22.5g环氧氯丙烷，分次滴加20mL50&nbsp;%的NaOH溶液，Ih内滴加完毕。一定搅拌下固化3h后再进行冷凝，控制冷却速度2°C/min至温度冷却至10°C以下。多次清洗后制备出粒径为100-300目的琼脂糖凝胶微球。

[0041 ] 实施例2

[0042] 取1g琼脂糖于250mL水中(琼脂糖水溶液浓度为4%)，常温搅拌lh，加入0.2gKCl继续搅拌0.5h后，加热至琼脂糖沸腾，溶液呈现透明状。准备1.75L甲苯溶液，加入30.45g聚醋酸乙烯酯和3.045g油酸。将有机相于70°C下恒温搅拌至混合均匀，将沸腾的琼脂糖水溶液倒入有机相中，一定搅拌下琼脂糖的分散乳化lh。一次性加入30g环氧氯丙烷，分次滴加25mL50&nbsp;%的NaOH溶液，Ih内滴加完毕。一定搅拌下固化3h后再进行冷凝，控制冷却速度2 °C/min至温度冷却至10°C以下。多次清洗后制备出粒径为100-300目的琼脂糖凝胶微球。

[0043] 实施例3

[0044]取15g琼脂糖于250mL水中(琼脂糖水溶液浓度为6% )，常温搅拌lh，加入

0.375gKCl继续搅拌0.5h后，加热至琼脂糖沸腾，溶液呈现透明状。准备1.625L甲苯溶液，加入35.3g聚醋酸乙烯酯和3.53g油酸。将有机相于70°C下恒温搅拌至混合均匀，将沸腾的琼脂糖水溶液倒入有机相中，一定搅拌下琼脂糖的分散乳化lh。一次性加入45g环氧氯丙烷，分次滴加38mL50&nbsp;%的NaOH溶液，Ih内滴加完毕。一定搅拌下固化3h后再进行冷凝，控制冷却速度2°C/min至温度冷却至10°C以下。多次清洗后制备出粒径为100-300目的琼脂糖凝胶微球。

[0045] 实施例4

[0046] 取20g琼脂糖于250mL水中(琼脂糖水溶液浓度为8% )，常温搅拌lh，加入0.36gKCl继续搅拌0.5h后，加热至琼脂糖沸腾，溶液呈现透明状。准备1.7L甲苯溶液，加入32.5g聚醋酸乙烯酯和3.25g油酸。将有机相于70°C下恒温搅拌至混合均匀，将沸腾的琼脂糖水溶液倒入有机相中，一定搅拌下琼脂糖的分散乳化lh。一次性加入60g环氧氯丙烷，分次滴加50mL50&nbsp;%的NaOH溶液，Ih内滴加完毕。一定搅拌下固化3h后再进行冷凝，控制冷却速度2 °C/min至温度冷却至10°C以下。多次清洗后制备出粒径为100-300目的琼脂糖凝胶微球。性能测试

Method A: This method was containing two steps. In the first step, iron oxide nanoparticles were synthesized according to procedures reported in literature [22,23], in which Fe(II) and Fe(III) are co-precipitated in an alkaline solution. The particles were then separated, washed and dried in an oven. In the second step, the nanoparticles were added to a stirring 4% agarose solution at 80 ◦C. The solution was then cooled to 50 ◦C and a 4% (v/v) span85 in cyclohexane solution was added to the mixture under vigorous stirring [21]. Spherical agarose particles were produced containing several nano-magnetic cores.

Method B: It was a one-pot process in which preparation of iron oxide nanoparticles and their coating by agarose were performed simultaneously. For this purpose, common methods for the preparation of iron oxide nanoparticles were used [22,23], except that the alkaline solution was replaced by an alkaline agarose solution.



Briefly, 0.2 g FeCl3 and 0.073 g FeCl2 were dissolved in 1 mol L−1 HCL (12 mL). Then, 0.05 g agarose was dissolved in 2 mol L−1 NaOH (25 mL) under heating to 80 ◦C. The alkaline agarose solution was deoxygenated by purging nitrogen and 2 mL of the Fe3+/Fe2+ solution was added to it, dropwise. The addition was made under nitrogen atmosphere at reflux conditions (90 ◦C) and magnetic stirring (800 rpm). The color of the bulk solution changed to black immediately, that indicates the production of Fe3O4 nanoparticles. By this method, magnetic micro-flake agarose particles were produced. The final mixture was cooled to ambient temperature and the prepared ACMNPs were separated by a permanent magnet and washed with water and 50% (v/v) water/ethanol, respectively, and stored under 20% (v/v) ethanol in 4 ◦C. 2.4. Activation and functionalization procedures for ACMNPs For partial crosslinking of the agarose particles and their functionalization by IDA ligand, an epichlorohydrin activation method was used as reported elsewhere [24]. After activation, 1 mL of the ACMNPs was added to 10 mL 0.1 mol L−1 ligand solution (pH 10, adjusted by 1 mol L−1 NaOH). The mixture was then shaken for 24 h at 30 ◦C. Finally, the particles were washed well with water and stored as a 20% v/v suspension under 20% (v/v) ethanol in 4 ◦C for later uses. Fig. 2 shows an schematic representation of the preparation procedure for ACMNPs and their activation and functionalization by IDA.