Direct Immobilization of Engineered Nanobodies on Gold Sensors

Single-domain antibodies, known as nanobodies, have great potential as biorecognition elements for sensors because of their small size, affinity, specificity, and robustness. However, facile and efficient methods of nanobody immobilization are sought that retain their maximum functionality. Herein, we describe the direct immobilization of nanobodies on gold sensors by exploiting a modified cysteine strategically positioned at the C-terminal end of the nanobody. The experimental data based on secondary ion mass spectrometry, circular dichroism, and surface plasmon resonance, taken together with a detailed computational work (molecular dynamics simulations), support the formation of stable and well-oriented nanobody monolayers. Furthermore, the nanobody structure and activity is preserved, wherein the nanobody is immobilized at a high density (approximately 1 nanobody per 13 nm(2)). The strategy for the spontaneous nanobody self-assembly is simple and effective and possesses exceptional potential to be used in numerous sensing platforms, ranging from clinical diagnosis to environmental monitoring.

Automated summary

This study describes a method for directly immobilizing nanobodies on gold sensors through the introduction of a modified cysteine strategically placed at the C-terminal end of the nanobody. Experimental data and computational simulations support the formation of stable and well-oriented nanobody monolayers, allowing for high-density immobilization while preserving the nanobody's structure and activity. The strategy of spontaneous nanobody self-assembly is simple and effective, with potential applications in various sensing platforms from clinical diagnostics to environmental monitoring.