Impedance biosensors have been widely studied for applications to biomedicine, environmental monitoring, food, and agriculture.  The most common format for AC impedance biosensors involves surface immobilization of an antibody, receptor protein, DNA strand, or other species capable of bio-recognition, and AC impedance detection of the binding event. 

One obstacle to application of impedance biosensors is the stability and reproducibility of protein immobilization. Alkane-thiols deposited on gold (Au) electrodes or alkenes deposited on silicon (Si) electrodes were used as impedance biosensors previously. However, Au-thiol bonds are unstable, so proteins are only stable atop such interfaces for days to weeks. While Si sensor electrodes are more stable, gradual oxidation of Si eventually displaces the polymer-protein film. Ti/TiO₂ is an attractive alternative substrate material for impedance biosensors due to the biocompatibility of Ti, and the self-limiting thickness of TiO₂. 

Sputtered Ti films are first cathodically stripped of the Ti native oxide, and then galvanostatically anodized in 1.0 M H₂SO₄.  Following anodization, nitrogen doping is accomplished by annealing at 600°C in NH₃/N₂ in a tube furnace.  A bifunctional reagent was synthesized with a carboxylate group at one end, and a phosphonate group at the other terminal end that could bind to hydroxyl groups on the surface of TiO2.  This reagent is used to attach mouse monoclonal antibodies to the TiO₂ surface though amide bond formation using EDC/NHSS. Peanut protein Ara h1, an allergenic food protein, can then be detected at the Ti/TiO₂ sensor electrodes. The response as a function of Ara h1 concentration follows the Langmuir adsorption isotherm, as expected.