开发高性能磁珠特异性富集核糖核酸(RNA)在生物医学研究领域具有重要意义。在这里,提出了一种简单的策略来制造硼酸酯修饰的聚乙烯亚胺接枝的磁性琼脂糖珠(BPMAB),它可以通过硼酸酯亲和力选择性分离含顺式二醇的物质。通过高速剪切机乳化油包水乳液,控制碱性磁性琼脂糖珠的大小,提高了BPMAB的比表面积。随后,为了修饰更多的硼酸配体,接枝了具有优异亲水性和许多反应位点的支链PEI。共价固定2,4-二氟-3-甲酰基苯基硼酸(2,4-DFPBA),用于在生理条件(pH7.4)下选择性捕获含顺式二醇的物质。直径范围从1.86μm到11.60μm的BPMAB具有明显的球形结构,在水溶液中具有优异的磁响应性和悬浮能力。β-烟酰胺腺嘌呤二核苷酸(β-NAD),短链顺式二醇携带剂,选择BPMAB作为目标分子进行吸附性能评价,BPMAB对β-NAD的最大吸附量可达205.11mgg-1。此外,BPMAB作为吸附剂用于从哺乳动物细胞中选择性富集RNA。BPMAB对RNA的最大吸附容量为140.50mgg-1。在优化条件下,基于BPMAB的MSPE成功富集了高质量的总RNA,其核糖体RNA的28S与18S的比值范围为2.06至2.16.根据GADPH基因的PCR分析,提取的总RNA成功逆转录成cDNA。因此,我们认为,基于BPMAB的MSPE可能适用于复杂生物系统中RNA的特异性富集。
Exploiting high-performance magnetic beads for specific enrichment of ribonucleic acid (RNA) has important significance in the biomedical research field. Herein, a simple strategy was proposed for fabricating boronate-decorated polyethyleneimine-grafted magnetic agarose beads (BPMAB), which can selectively isolate cis-diol-containing substances through boronate affinity. The size of the basic magnetic agarose beads was controlled through the emulsification of the water-in-oil emulsion with a high-speed shear machine, which enhanced the specific surface area of BPMAB. Subsequently, to modify more boronic acid ligands, branched PEI with excellent hydrophilicity and numerous reaction sites was grafted. 2,4-Difluoro-3-formylphenyl boronic acid (2,4-DFPBA) was covalently immobilized for selectively capturing cis-diol-containing substances under physiological condition (pH 7.4). The BPMAB with a diameter range from 1.86 μm to 11.60 μm possessed clearly spherical structure, and excellent magnetic responsiveness and suspension ability in aqueous solution. β-Nicotinamide adenine dinucleotide (β-NAD), a short-chain cis-diol carrying agent, was selected as a target molecule for evaluating the adsorption property of BPMAB and the maximum adsorption capacity of BPMAB for β-NAD could reach 205.11 mg g-1. In addition, the BPMAB as adsorbent was used to selectively enrich RNA from mammalian cells. The maximum adsorption capacity of BPMAB for RNA was 140.50 mg g-1. Under optimized conditions, the BPMAB-based MSPE successfully enriched the high-quality total RNA with 28S to 18S ribosomal RNA ratios ranging from 2.06 to 2.16. According to the PCR analysis of GADPH gene, the extracted total RNA was successfully reverse transcribed into cDNA. Therefore, we believe that the BPMAB-based MSPE could be applicable for the specific enrichment of RNA from complex biological systems.