Oriented immobilization of a delicate glucose-sensing protein on silica nanoparticles.

Silica nanoparticles are widely used platform materials for the immobilization of proteins to realize applications in biomedicine and biotechnology. We here report on the use of a highly delicate protein for the systematic evaluation of routes for the surface modification of multifunctional silica nanoparticles. To investigate how surface immobilization methods affect the functionality of surface-bound proteins, we constructed a novel fusion protein, dubbed FlipHOB, that combines the glucose sensor protein FLIP with a variant of the commercially-available self-ligating Halo-tag. As indicated by the spectroscopic properties and sensing capabilities of FlipHOB, the oriented immobilization of this protein through its HOB tag domain or DNA-directed immobilization were superior over the non-directional statistical immobilization via glutardialdehyde-mediated cross-coupling. Immobilization through double-stranded DNA bridges also allows for the triggered disassembly of FlipHOB nanosensors and the controlled recovery of the sensor protein. We demonstrate that the nanosensors are functional in in vitro settings and can be used for imaging in vivo. We believe that our results show generic strategies and provide essential guidelines for the development of protein-based nanoparticle sensors for applications in the life sciences.