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Process for the specific immobilisation of olfactory receptors on gold electrodes and microelectrodes and its monitoring by electrochemical impedance

A new technique for specific immobilization of nanosomes containing ORs (and other GPCRs) on gold electrodes and microelectrodes has been developed based on a new mixed self-assembled-monolayer technique

In order to develop a biosensor based on the electrical properties of the olfactory receptors a first necessary step consists on the specific immobilization of the ORs on a measuring electrode. The immobilization strategy depends very much on the measuring magnitude used to monitor the sensing action. Of the various existing possibilities we finally decided to perform electrochemical impedance measurements.

The reasons to chose such a measuring principle were that it naturally allows performing measurements in physiological medium necessary to keep the functionality of the receptors and that the geometrical configuration is well suited for immobilization purposes (the sensing biological sample has to be immobliized essentially on a flat conducting substrate). A main drawback of this technique is that it requires a previous passivation of the gold substrate in order to minimize as much as possible the presence of leakage currents. However, the same pasivation layer can be used for specific immobilization purposes thus making a double function.

A new technique for immobilization of rat OR I7 (and other GPCRs in general) is based on mixed SAMs (self-assembled monolayer) by introduction of biotine groups in a thiol SAM grafted on gold surface, which allows to strongly attaching biotinylated antibody through biotin-neutravidin bond and then to recognise and bind selectively specific OR or in general other GPCRs.

The monitoring of the immobilization process step by step has been performed by different techniques Electrochemical Impedance Spectroscopy in order to provide a firm basis on the efectiviness and reproducibility of this essential technique.

Nyquist plots of impedance spectra have been taken in PBS without redox couple in the frequency range from 500mHz to 100kHz. The impedance data were fitted with commercially available software Zplot/Zview (Scibner Associates Inc.). The equivalent circuit was found to fit adequately the data over the entire frequency range.

The circuit follows a standard Randles cell and includes the following four elements:
- The ohmic resistance of the electrolyte solution, Rs.
- The constant phase element impedance, ZCPE1.
- The generalised finite Warburg impedance in the open circuit model, ZW1o.
- The polarisation resistance, Rp.

Starting from the bare gold, the deposition of the mixed SAM monolayer is found to produce a significant increase of the polarisation resistance together with a significant decrease of the double layer capacitance which confirms the good insulating properties of the mixed monolayer already found in the CV characterisation. In all the successive steps we found a systematic decrease of the polarisation resistance and of the Warburg resistance, which indicate a recovery in the efficiency of the mass transfer phenomenon and/or difference in the dielectric or conductive properties of the electrode surface with formation of new layer. The value of the solution resistance is found to remain practically constant. The deviation of CPE1-P and W0-P from the ideal values 1 and 0.5, respectively, is taken as a measure of the presence of spurious effects like the roughness of the electrode surface and some anomalies of the mass transfer to follow the standard diffusion equation.

Results show that the polarization resistance is a suitable parameter to monitor the process.

We demonstrated that the EIS technique can not only be used to monitor the step by step formation of the immobilization layer but it can also be used to monitor the concentration of immobilized olfactory receptor (or other GPCRs). Results have been verified for bovine rhodopsin (not discussed here) and for rat OR I7.

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