Biomolecule Design Group, Institute for Materials Research (IMO), Hasselt University, Diepenbeek BE-3590, Belgium.
Faculty of Food Technology and Biotechnology, Can Tho University of Technology, Can Tho 900000, Vietnam.
Biosensors (Basel). 2016 Jul 5;6(3):34. doi: 10.3390/bios6030034.
Surface bioconjugation of biomolecules has gained enormous attention for developing advanced biomaterials including biosensors. While conventional immobilization (by physisorption or covalent couplings using the functional groups of the endogenous amino acids) usually results in surfaces with low activity, reproducibility and reusability, the application of methods that allow for a covalent and uniformly oriented coupling can circumvent these limitations. In this study, the nanobody targeting Vascular Cell Adhesion Molecule-1 (NbVCAM1), an atherosclerotic biomarker, is engineered with a C-terminal alkyne function via Expressed Protein Ligation (EPL). Conjugation of this nanobody to azidified silicon wafers and Biacore™ C1 sensor chips is achieved via Copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) "click" chemistry to detect VCAM1 binding via ellipsometry and surface plasmon resonance (SPR), respectively. The resulting surfaces, covered with uniformly oriented nanobodies, clearly show an increased antigen binding affinity, sensitivity, detection limit, quantitation limit and reusability as compared to surfaces prepared by random conjugation. These findings demonstrate the added value of a combined EPL and CuAAC approach as it results in strong control over the surface orientation of the nanobodies and an improved detecting power of their targets-a must for the development of advanced miniaturized, multi-biomarker biosensor platforms.
生物分子的表面生物缀合在开发先进的生物材料方面引起了广泛关注，包括生物传感器。虽然传统的固定化方法（通过物理吸附或利用内源性氨基酸的官能团进行共价偶联）通常导致表面活性、重现性和可重复使用性较低，但应用允许共价和均匀定向偶联的方法可以避免这些限制。在这项研究中，通过表达蛋白连接（Expressed Protein Ligation，EPL）在靶向血管细胞黏附分子-1（Vascular Cell Adhesion Molecule-1，VCAM1）的纳米抗体的 C 末端引入炔基官能团。通过铜（I）催化的叠氮-炔环加成（Copper(I)-catalyzed azide-alkyne cycloaddition，CuAAC）“点击”化学将这种纳米抗体偶联到叠氮化硅晶片和 Biacore™ C1 传感器芯片上，分别通过椭偏术和表面等离子体共振（surface plasmon resonance，SPR）来检测 VCAM1 的结合。与通过随机偶联制备的表面相比，覆盖着均匀定向纳米抗体的这些表面明显显示出更高的抗原结合亲和力、灵敏度、检测限、定量限和可重复使用性。这些发现证明了 EPL 和 CuAAC 联合方法的附加值，因为它可以对纳米抗体的表面取向进行强有力的控制，并提高其目标的检测能力——这是开发先进的小型化、多生物标志物生物传感器平台的必要条件。
