客户采用我司硅氨基磁珠构建基于多巴胺的蛋白质模板分子印迹

On-line monitoring of the dopamine-based molecular imprinting processes for protein templates with the assistance of a fluorescent indicator

借助荧光指示剂在线监测基于多巴胺的蛋白质模板分子印迹过程
Yibin Liu, Huaisyuan Xie, Xinyi Li, Ying Sun, Zhiwei Zhu & Meiping Zhao 
Microchimica Acta volume 189, Article number: 138 (2022) Cite this article


Abstract
On-line monitoring of the dopamine (DA)-based molecular imprinting processes over Fe3O4@SiO2-NH2 nanoparticles (SiMNPs) is reported by using a real-time quantitative PCR machine. Taking advantages of the efficient fluorescence quenching capability of polydopamine (PDA) and its high binding affinity to rhodamine B (RhB), we performed molecular imprinting against different proteins with free dopamine as the functional monomer and RhB as a fluorescent indicator. Along with the template molecules, the fluorescent indicators were continuously encapsulated into the PDA layer formed on the surface of the SiMNPs, resulting in immediate quenching of the fluorescence, which can be conveniently monitored in real time. As proteins showed sequence-dependent influences on the oxidation of dopamine and subsequent self-assembly on the surface of the SiMNPs, the observed fluorescence signals clearly indicated the polymerization progress in the presence of the template proteins, allowing precise control of the reaction time for different templates at a given initial concentration. The optimum end point of the reaction was found to be when 90 ± 3% of the templates had been encapsulated into the polymer, which offered the highest imprinting factor and selectivity. We applied the approach to prepare a primary PDA-based surface imprinted polymer for a multifunctional protein apurinic/apyrimidinic endonuclease/redox effector factor 1 (APE1). After further introduction of 3-hydroxyphenylboronic acid to the interfaces between APE1 and PDA, the resultant molecularly imprinted polymers (MIP-II) enabled quantitative isolation APE1 from cell lysate samples. The developed approach will be useful for the quantitative preparation of PDA-based MIPs for precious template proteins with limited input quantity. It is also applicable for further study on the effects of different proteins or peptides on the PDA formation reactions.

使用实时定量 PCR 仪在线监测 Fe3O4@SiO2-NH2 纳米颗粒 (SiMNPs) 上的基于多巴胺 (DA) 的分子印迹过程。利用聚多巴胺（PDA）的高效荧光猝灭能力及其对罗丹明B（RhB）的高结合亲和力，我们以游离多巴胺为功能单体，RhB为荧光指示剂对不同蛋白质进行了分子印迹。荧光指示剂与模板分子一起被连续封装在形成于 SiMNPs 表面的 PDA 层中，导致荧光立即猝灭，可以方便地实时监测。由于蛋白质对多巴胺的氧化和随后在 SiMNPs 表面的自组装表现出序列依赖性影响，观察到的荧光信号清楚地表明了在模板蛋白存在下的聚合过程，从而可以精确控制不同的反应时间给定初始浓度的模板。发现反应的最佳终点是当 90 ± 3% 的模板被封装到聚合物中时，这提供了最高的印迹因子和选择性。我们应用该方法制备用于多功能蛋白脱嘌呤/脱嘧啶核酸内切酶/氧化还原效应因子 1 (APE1) 的初级基于 PDA 的表面印迹聚合物。在将 3-羟基苯基硼酸进一步引入 APE1 和 PDA 之间的界面后，所得的分子印迹聚合物 (MIP-II) 能够从细胞裂解物样品中定量分离 APE1。所开发的方法可用于定量制备基于 PDA 的 MIP，用于有限输入量的珍贵模板蛋白。它也适用于进一步研究不同蛋白质或肽对PDA形成反应的影响。

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