Hydrogen peroxide sensors for cellular imaging based on horse radish peroxidase reconstituted on polymer-functionalized TiO2 nanorods

We describe the reconstitution of apo-horse radish peroxidase (apo-HRP) onto TiO2 nanorods functionalized with a multifunctional polymer. After functionalization, the horse radish peroxidase (HRP) functionalized TiO2 nanorods were well dispersible in aqueous solution, catalytically active and biocompatible, and they could be used to quantify and image H2O2 which is a harmful secondary product of cellular metabolism. The shape, size and structure of TiO2 nanorods (anatase) were analyzed by transmission electron microscopy (TEM), high resolution TEM (HRTEM), electron diffraction (ED) and X-ray diffraction (XRD). The surface functionalization, HRP reconstitution and catalytic activity were confirmed by UV-Vis, FT-IR, CLSM and atomic force microscopy (AFM). Biocompatibility and cellular internalization of active HRP reconstituted TiO2 nanorods were confirmed by a classical MTT cytotoxicity assay and confocal laser scanning microscopy (CLSM) imaging, respectively. The intracellular localization allowed H2O2 detection, imaging and quantification in HeLa cells. The polymer functionalized hybrid system creates a complete sensor including a cell positioning system'' in each single particle. The flexible synthetic concept with functionalization by post-polymerization modification allows introduction of various dyes for sensitisation at different wavelengths and introduction of various anchor groups for anchoring on different particles.