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首页> 外文期刊>Nano letters >Magnetic Separation of Elastin-like Polypeptide Receptors for Enrichment of Cellular and Molecular Targets
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Magnetic Separation of Elastin-like Polypeptide Receptors for Enrichment of Cellular and Molecular Targets

机译：用于富集细胞和分子靶标的弹性蛋白样多肽受体的磁性分离

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摘要

Protein-conjugated magnetic nanoparticles (mNPs) are promising tools for a variety of biomedical applications, from immunoassays and biosensors to theranostics and drug-delivery. In such applications, conjugation of affinity proteins (e.g., antibodies) to the nanoparticle surface many times compromises biological activity and specificity, leading to increased reagent consumption and decreased assay performance. To address this problem, we engineered a biomolecular magnetic separation system that eliminates the need to chemically modify nanoparticles with the capture biomolecules or synthetic polymers of any kind. The system consists of (i) thermoresponsive magnetic iron oxide nanoparticles displaying poly(N-isopropylacrylamide) (pNIPAm), and (ii) an elastin-like polypeptide (ELP) fused with the affinity protein Cohesin (Coh). Proper design of pNIPAm-mNPs and ELP-Coh allowed for efficient cross aggregation of the two distinct nanoparticle types under collapsing stimuli, which enabled magnetic separation of ELP-Coh aggregates bound to target Dockerin (Doc) molecules. Selective resolubilization of the ELP-Coh/Doc complexes was achieved under intermediate conditions under which only the pNIPAm-mNPs remained aggregated. We show that ELP-Coh is capable of magnetically separating and purifying nanomolar quantities of Doc as well as eukaryotic whole cells displaying the complementary Doc domain from diluted human plasma. This modular system provides magnetic enrichment and purification of captured molecular targets and eliminates the requirement of biofunctionalization of magnetic nanoparticles to achieve bioseparations. Our streamlined and simplified approach is amenable for point-of-use applications and brings the advantages of ELP-fusion proteins to the realm of magnetic particle separation systems.
机译：蛋白 - 缀合的磁性纳米颗粒（的MNP）是有前途的工具，用于多种生物医学应用，从免疫测定和生物传感器治疗诊断学和药物递送。在这种应用中，亲和蛋白（例如，抗体）至纳米粒子表面多次的缀合生物损害活性和特异性，导致增加的试剂消耗和降低的测定性能。为了解决这个问题，我们工程改造，从而无需进行化学改性的纳米粒子与生物分子捕获或任何种类的合成聚合物生物分子的磁分离系统。该系统由（i）的温敏磁性氧化铁纳米颗粒的聚显示（N-异丙基丙烯酰胺）（PNIPAM），和（ii）与所述亲和蛋白质黏结（COH）稠合的弹性蛋白样多肽（ELP）。 PNIPAM-的MNP和ELP-COH的适当设计允许用于两个不同的纳米粒子类型的有效的交叉聚合塌陷的刺激，这使结合到目标坞因子（DOC）分子ELP-COH聚集体的磁分离下。所述ELP-COH /文件复合物的选择性再增的混合物在其中仅PNIPAM-的MNP仍然聚集中间的条件下实现。我们表明，ELP-COH能够磁分离和纯化文件的纳摩尔数量以及显示从稀释的人血浆互补文件真核生物域的全细胞。这种模块化系统提供的捕获的分子靶磁性富集和纯化，并消除磁性纳米粒子以达到生物分离的生物功能化的要求。我们的简化和简化的方法是适合于点使用的应用程序，并带来ELP融合蛋白的优点，以磁性粒子的分离系统的领域。

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