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.