Contarino Mark R, Sergi Mauro, Harrington Adrian E, Lazareck Adam, Xu Jimmy, Chaiken Irwin
Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, PA 19102, USA.
J Mol Recognit. 2006 Jul-Aug;19(4):363-71. doi: 10.1002/jmr.783.
As part of an effort to develop nanoelectronic sensors for biological targets, we tested the potential to incorporate coiled coils as metallized, self-assembling, site-specific molecular linkers on carbon nanotubes (CNTs). Based on a previously conceived modular anchor-probe approach, a system was designed in which hydrophobic residues (valines and leucines) form the interface between the two helical peptide components. Charged residues (glutamates and arginines) on the borders of the hydrophobic interface increase peptide solubility, and provide stability and specificity for anchor-probe assembly. Two histidine residues oriented on the exposed hydrophilic exterior of each peptide were included as chelating sites for metal ions such as cobalt. Cysteines were incorporated at the peptide termini for oriented, thiol-mediated coupling to surface plasmon resonance (SPR) biosensor surfaces, gold nanoparticles or CNT substrates. The two peptides were produced by solid phase peptide synthesis using Fmoc chemistry: an acidic 42-residue peptide E42C, and its counterpart in the heterodimer, a basic 39-residue peptide R39C. The ability of E42C and R39C to bind cobalt was demonstrated by immobilized metal affinity chromatography and isothermal titration calorimetry. SPR biosensor kinetic analysis of dimer assembly revealed apparent sub-nanomolar affinities in buffers with and without 1 mM CoCl2 using two different reference surfaces. For device-oriented CNT immobilization, R39C was covalently anchored to CNT tips via a C-terminal cysteine residue. Scanning electron microscopy was used to visualize the assembly of probe peptide (E42C) N-terminally labeled with 15 nm gold nanoparticles, when added to the R39C-CNT surface. The results obtained open the way to develop CNT tip-directed recognition surfaces, using recombinant and chemically synthesized chimeras containing binding epitopes fused to the E42C sequence domain.
作为开发用于生物靶点的纳米电子传感器工作的一部分，我们测试了将卷曲螺旋作为金属化、自组装、位点特异性分子连接体整合到碳纳米管（CNT）上的潜力。基于先前设想的模块化锚定-探针方法，设计了一个系统，其中疏水残基（缬氨酸和亮氨酸）形成两个螺旋肽组分之间的界面。疏水界面边界上的带电残基（谷氨酸和精氨酸）增加了肽的溶解度，并为锚定-探针组装提供稳定性和特异性。每个肽暴露的亲水性外部上定向的两个组氨酸残基被用作钴等金属离子的螯合位点。在肽末端引入半胱氨酸用于通过硫醇介导定向偶联到表面等离子体共振（SPR）生物传感器表面、金纳米颗粒或CNT底物上。这两种肽通过使用Fmoc化学的固相肽合成制备：一种酸性的42个残基的肽E42C，以及其在异二聚体中的对应物，一种碱性的39个残基的肽R39C。通过固定化金属亲和色谱和等温滴定量热法证明了E42C和R39C结合钴的能力。使用两个不同的参考表面，对二聚体组装进行的SPR生物传感器动力学分析显示，在有和没有1 mM CoCl2的缓冲液中，亲和力明显为亚纳摩尔级。对于面向器件的CNT固定化，R39C通过C末端半胱氨酸残基共价锚定到CNT尖端。当将用15 nm金纳米颗粒N末端标记的探针肽（E42C）添加到R39C-CNT表面时，使用扫描电子显微镜观察其组装情况。所获得的结果为利用含有与E42C序列域融合的结合表位的重组和化学合成嵌合体开发CNT尖端定向识别表面开辟了道路。
