We report here the results of human olfactory receptor (OR) 17-40 stimulation with some odorants probed by means of the double-channel surface plasmon resonance platform NanoSPR-6. OR 17-40 tagged with N-terminal cmyc sequence was heterologously co-expressed with Galpha(olf) protein in yeast, and receptor-carrying nanosomes were prepared from yeast membrane fraction. Then, receptors were specifically captured via anti-cmyc antibody attached to the gold-coated substrate in orientated or random way. Measurement of odorants effects were carried out in the presence of GTP-gamma-S in differential mode in order to compensate bulk changes of refractive index. For the first time, biosensing efficiency of olfactory films was discussed in terms of their thickness and Galpha(olf) accessibility to GTP-gamma-S. Bell-shaped response profile with two maxima (near 1 nM and near 1 microM) was established for helional, which is documented as highly specific agonist of OR 17-40. Unrelated odorant heptanal used as control, did not evoke significant variations of differential signal.
Stable surface functionalization is required in many applications such as in biosensing. For this purpose, hydrocarbon‐based plasma polymer films (PPFs) were investigated by controlling cross‐linking and film structure at the nanoscale, while functional groups were incorporated using a CO2/C2H4 low pressure plasma. In particular, an oxygen‐rich termination layer of 1 nm (top layer) was gradually deposited onto a more cross‐linked PPF (base layer) with varying thickness (<20 nm) yielding highly stable yet functional a‐C:H:O films as compared to conventional oxygen‐rich PPFs. Such stabilized nano‐layers provided a homogeneous functionalization of Au‐coated (flat) biochips as used for immunosensing as well as of nanostructured Au‐coated surfaces explored for enhanced biosensing. The used periodic grating coated by the highly stable a‐C:H:O nano‐layer can be adjusted to the required environmental refractive index range yielding a distinct sensitivity enhancement for biosensing.
The increase of the sensitivity of surface plasmon resonance (SPR) refractometers was studied experimentally by forming a periodic relief in the form of a grating with submicron period on the surface of the Au-coated chip. Periodic reliefs of different depths and spatial frequency were formed on the Au film surface using interference lithography and vacuum chalcogenide photoresists. Spatial frequencies of the grating were selected close to the conditions of Bragg reflection of plasmons for the working wavelength of the SPR refractometer and the used environment (solution of glycerol in water). It was found that the degree of refractometer sensitivity enhancement and the value of the interval of environment refractive index variation, Δn, in which this enhancement is observed, depend on the depth of the grating relief. By increasing the depth of relief from 13.5 ± 2 nm to 21.0 ± 2 nm, Δn decreased from 0.009 to 0.0031, whereas sensitivity increased from 110 deg./RIU (refractive index unit) for a standard chip up to 264 and 484 deg./RIU for the nanostructured chips, respectively. Finally, it was shown that the working range of the sensor can be adjusted to the refractive index of the studied environment by changing the spatial frequency of the grating, by modification of the chip surface or by rotation of the chip.
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