客戶采用我司硅氨基磁珠構建基于多巴胺的蛋白質模板分子印跡
客戶采用我司硅氨基磁珠構建基于多巴胺的蛋白質模板分子印跡
On-line monitoring of the dopamine-based molecular imprinting processes for protein templates with the assistance of a fluorescent indicator
借助熒光指示劑在線監(jiān)測基于多巴胺的蛋白質模板分子印跡過程
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.
使用實時定量 PCR 儀在線監(jiān)測 Fe3O4@SiO2-NH2 納米顆粒 (SiMNPs) 上的基于多巴胺 (DA) 的分子印跡過程。利用聚多巴胺（PDA）的高效熒光猝滅能力及其對羅丹明B（RhB）的高結合親和力，我們以游離多巴胺為功能單體，RhB為熒光指示劑對不同蛋白質進行了分子印跡。熒光指示劑與模板分子一起被連續(xù)封裝在形成于 SiMNPs 表面的 PDA 層中，導致熒光立即猝滅，可以方便地實時監(jiān)測。由于蛋白質對多巴胺的氧化和隨后在 SiMNPs 表面的自組裝表現出序列依賴性影響，觀察到的熒光信號清楚地表明了在模板蛋白存在下的聚合過程，從而可以精確控制不同的反應時間給定初始濃度的模板。發(fā)現反應的最佳終點是當 90?±?3% 的模板被封裝到聚合物中時，這提供了最高的印跡因子和選擇性。我們應用該方法制備用于多功能蛋白脫嘌呤/脫嘧啶核酸內切酶/氧化還原效應因子 1 (APE1) 的初級基于 PDA 的表面印跡聚合物。在將 3-羥基苯基硼酸進一步引入 APE1 和 PDA 之間的界面后，所得的分子印跡聚合物 (MIP-II) 能夠從細胞裂解物樣品中定量分離 APE1。所開發(fā)的方法可用于定量制備基于 PDA 的 MIP，用于有限輸入量的珍貴模板蛋白。它也適用于進一步研究不同蛋白質或肽對PDA形成反應的影響。
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