Diamond micro-cantilevers as transducers for Olfactory Receptors - based biosensors

Introduction Olfactory receptors (ORs) are proteins located in the plasma membrane of olfactory sensory neurons. They belong to the family of G protein-coupled receptors. Individual ORs bind various odorants with distinct... [ view full abstract ]

Introduction
Olfactory receptors (ORs) are proteins located in the plasma membrane of olfactory sensory neurons. They belong to the family of G protein-coupled receptors. Individual ORs bind various odorants with distinct affinities and specificities. Therefore, ORs combined into arrays are considered as highly promising for the conception of biosensors for odorant detection. Here we investigated a new approach consisting of chemically grafting the ORs onto synthetic diamond grown by Chemical Vapor Deposition. Because of its stability, biocompatibility, and ability to immobilize biological targets, diamond is a material of choice for biosensors.
Methods
ORs were grafted onto diamond surfaces using two routes, either (1) through covalent bonding of hexanoic acid radical on diamond followed by EDC/NHS peptidic coupling to an OR, or (2) using covalent attachment of nitriloacetic acid (NTA) on diamond as chelating agent, which binds an 6His-tagged OR via NTA-Ni interaction. Both grafting procedures were validated by electrochemical impedance spectroscopy (EIS) on boron doped diamond electrodes. They were then applied to bulk diamond micro-cantilevers freshly hydrogenated in hydrogen plasma in a Chemical Vapour Deposition reactor. Functionality for odorant detection was assessed in the liquid phase using a Laser Doppler read-out system.
Results
OR7D4 was grafted through route (1), while 6His-tagged M71 was grafted through route (2). The Nyquist plots obtained from both EIS spectra exhibit an increase of the charge-transfer resistance, thus of the electrode coverage, showing for the first time an efficient immobilization of ORs on diamond.
Then, the 6His-M71 micro-cantilever sensor exhibited a good sensitivity to its known odorant ligand acetophenone, with a frequency shift near 100Hz for 1μM exposure (320Hz for 10μM), and a good selectivity against a non-ligand odorant. Likewise, the OR7D4 micro-cantilever sensor showed a sensitivity of 200Hz for exposure to its ligand androstenone at 1µM with a good selectivity against non-ligand odorants.
Discussion
Micro-cantilevers grafted with ORs are thus functional, and yield a quantitative, reproducible response to target analytes, at least at the micromolar level, and selectivity among odorants. The results are promising for the development of bioelectronic noses consisting of arrays of OR-grafted diamond-based resonators for specifically detecting target odorants.