Department of Mechanical Engineering, University of California, Santa Barbara, CA 93106, USA.
Proc Natl Acad Sci U S A. 2011 Apr 26;108(17):6909-14. doi: 10.1073/pnas.1014753108. Epub 2011 Apr 12.
Affinity reagents that bind to specific molecular targets are an essential tool for both diagnostics and targeted therapeutics. There is a particular need for advanced technologies for the generation of reagents that specifically target cell-surface markers, because transmembrane proteins are notoriously difficult to express in recombinant form. We have previously shown that microfluidics offers many advantages for generating affinity reagents against purified protein targets, and we have now significantly extended this approach to achieve successful in vitro selection of T7 phage-displayed peptides that recognize markers expressed on live, adherent cells within a microfluidic channel. As a model, we have targeted neuropilin-1 (NRP-1), a membrane-bound receptor expressed at the surface of human prostate carcinoma cells that plays central roles in angiogenesis, cell migration, and invasion. We show that, compared to conventional biopanning methods, microfluidic selection enables more efficient discovery of peptides with higher affinity and specificity by providing controllable and reproducible means for applying stringent selection conditions against minimal amounts of target cells without loss. Using our microfluidic system, we isolate peptide sequences with superior binding affinity and specificity relative to the well known NRP-1-binding RPARPAR peptide. As such microfluidic systems can be used with a wide range of biocombinatorial libraries and tissue types, we believe that our method represents an effective approach toward efficient biomarker discovery from patient samples.
亲和试剂能与特定的分子靶标结合，是诊断和靶向治疗的重要工具。特别需要先进的技术来生成针对细胞表面标志物的试剂，因为跨膜蛋白很难以重组形式表达。我们之前已经证明，微流控技术在生成针对纯化蛋白靶标的亲和试剂方面具有许多优势，现在我们已经大大扩展了这种方法，成功地在体外选择了能够识别微流控通道内活贴壁细胞表面标志物的 T7 噬菌体展示肽。作为模型，我们针对神经纤毛蛋白-1（NRP-1），这是一种在人前列腺癌细胞表面表达的膜结合受体，在血管生成、细胞迁移和侵袭中发挥核心作用。我们发现，与传统的生物淘选方法相比，微流控选择通过提供可控和可重复的方法，以最小的靶细胞量施加严格的选择条件，而不会损失，从而能够更有效地发现具有更高亲和力和特异性的肽。使用我们的微流控系统，我们分离到的肽序列相对于众所周知的 NRP-1 结合肽 RPARPAR 具有更高的结合亲和力和特异性。由于这种微流控系统可以与广泛的生物组合文库和组织类型一起使用，我们相信我们的方法代表了一种从患者样本中高效发现生物标志物的有效方法。
