London Centre for Nanotechnology, University College London, London, UK.
Department of Biochemical Engineering, University College London, London, UK.
Nanoscale. 2024 Oct 31;16(42):19881-19896. doi: 10.1039/d4nr02762k.
The ease of expression and engineering of single domain antibodies, known as nanobodies, make them attractive alternatives to conventional antibodies in point-of-care diagnostics such as lateral flow assays. In lateral flow assays, gold nanoparticle bioconjugates serve as labels which display affinity molecules on the gold surface. While examples of nanobody gold nanoparticle bioconjugates exist, few utilise the simple one-step approach of physisorption owing to undesirable nanoparticle aggregation and loss of functionality. Here we show that engineering nanobodies into multivalent structures can significantly enhance their functionality when physisorbed onto gold nanoparticles. This approach enables resulting bioconjugates to withstand multiple processing steps required for long-term nanoparticle storage within lateral flow assays. Specifically, we show that the trivalent version of VHHV nanobody (VHH3) against the S1 protein of SARS-CoV-2 can be immobilised onto gold nanoparticles through passive adsorption. Unlike its monovalent and bivalent nanobody counterparts, using VHHV3 preserves nanoparticle stability under salt stress, blocking, washing, and freeze-drying conditions while maintaining picomolar sensitivity to the S1 protein. We anticipate that this facile strategy is a significant advancement towards the integration of nanobodies in lateral flow assay development.
单域抗体（即纳米抗体）表达和工程改造相对容易，在即时检测（如侧向流检测）等领域中，它们作为传统抗体的替代物具有很大吸引力。在侧向流检测中，金纳米颗粒生物缀合物可用作标签，将亲和分子显示在金表面。虽然已有纳米抗体-金纳米颗粒生物缀合物的实例，但由于纳米颗粒聚集和功能丧失等不理想的问题，很少采用简单的物理吸附一步法。在这里，我们展示了将纳米抗体工程化为多价结构，可以显著增强其在物理吸附到金纳米颗粒上时的功能。这种方法使得到的生物缀合物能够承受侧向流检测中用于长期纳米颗粒储存的多个处理步骤。具体来说，我们展示了针对 SARS-CoV-2 S1 蛋白的 VHHV 纳米抗体（VHH3）的三价形式可以通过被动吸附固定到金纳米颗粒上。与单价和二价纳米抗体相比，使用 VHH3 可以在盐胁迫、阻断、洗涤和冻干条件下保持纳米颗粒的稳定性，同时保持对 S1 蛋白的皮摩尔灵敏度。我们预计，这种简单的策略是将纳米抗体整合到侧向流检测开发中的重大进展。
