Comparison between in-vitro and in-vivo studies

The comparison of an artificial nanosensor-system based on a rGO field-effect transistor with a natural insect antenna has to be interpreted with much care. The natural system is stimulated by an odor stimulus in air consists of odorants reach the sensillar lymph through a hydrophobic chitin cuticle containing waxy pore kettles. Entered odorants is selectively binding and carrying by the soluble odorant-binding proteins (OBP), forming an odorant-OBP complex and transport to a specific membrane bound odorant receptors for processing the signal by opening of ion channels in the membrane of the sensory neuron (Figure 4-5A) (in-vivo).

In contrast, the electronic nanosensor-system is stimulated by an odorant molecules dissolved in water that delivered in a micro-flow cell, consists of OBPs immobilized to a Figure 4-13. In-vivo antennal dose-response relation to OBP occupancy. EAG measurements of the responses to a dilution series of 6-methyl-5-heptene-2-one in air: the EAG response increases with the odorant concentration. Error bars show standard deviations from 10 repeats.

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graphene gate of a FET for further electric conduction process by opening the gate channel (in-vitro). However, this artificial system is partially demonstrate about the natural system of insect olfaction and ideal for comparison of olfactory information process up to OBP level.

Additionally, a more detailed comparison might only be useful if the OBP utilized for the experiment is one with a major contribution to the sum signal of the whole insect antenna (EAG response).

For a first step of a more detailed comparison, the stimulus quantity has to be aligned.

Henry’s law provides a relation between the concentration of a compound in an aqueous solution, ca, and its vapor pressure, p:

ca = H^cp . p

As an example, we compare the situation for 6-methyl-5-hepten-2-one for which Henry’s law constant, is known:

H^cp=0.14 Mol m^-3 Pa^-1

With the measured range of p*1/2 = 6-80 μbar we obtain an equivalent 6-methyl-5-hepten-2-one concentration in the sensillum lymph for which half of the EAG-response is measured of c1/2 = 0.09 - 1.2 mM. Using Hcp = 0.31 Mol m^-3 Pa^-1 of 3-octanol and the measured range of p*1/2 = 0.7-7 μbar we obtain an equivalent 3-octanol concentration in the sensillum lymph for which half of the EAG-response is measured of c1/2 = 0.02 – 0.2 mM.

Our lowest concentration of 3-octanol in the flow cell was 300 μM which makes sense given the Kd= 3 mM, but it could have been much lower (certainly measurements are possible with 30 μM, corresponding to a partial pressure of 1 μbar, cf. simulations displayed in Figure 4-9B).

Hence, the dilutions measured by natural and artificial olfaction systems are in a similar range of magnitude and these two techniques have shown comparable sensitivity to the tested stimuli, though artificial system partially mimic the natural system. However, the natural system shows a much quicker response yielding times at the milli-seconds level and recovery times at the seconds level in comparison to minutes-level in the artificial system. This hints at the fact that both binding and removal of the ligand and exhibiting olfactory response in the

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natural systems are significantly quicker due to involvement of several olfactory components.

For instance, comparing the binding constants of the OBPs in both experiments shows one to two orders of magnitudes higher values for kon and for koff in the EAG measurements.

The artificial system might display a delayed response because of immobilization effects delaying the approach of odorants to the tested OBPs. However, the same immobilizing strategy was used for OBP14 of A. mellifera yielding significantly higher kon for selected ligands, rendering immobilization effects might be one of explanation for the different response times of the artificial system unlikely (Larisika et al. 2015). Unfortunately, there are no data about rate constants for all the components of the natural T. castaneum system available. Thus, we can speculate that the fact of the kon of the natural system being much quicker, might be due to additional phase-transfer catalytic process (22) employed during the delivery of the hydrophobic odorants from the external lipid layer through waxy pore kettles to the sensillum lymph (Sharma et al. 2015). Because of the low water solubility of many semiochemicals, transfer odorants from sensillum lymph to respective odorant receptor by OBPs may be a rate limiting step in the artificial system. The fact that both, the artificial and the natural system display similar Kd, but showing equally increased kinetic constants for association and dissociation in the natural system is highly suggesting the involvement of a catalytic process, which accelerates both, back- and forth-reaction.

4.5 Conclusion

We have demonstrated the detection of specific odorant molecules using an artificial olfactory biosensor based on reduced graphene oxide-field effect transistor (rGO-FET) functionalized with odorant binidng protein TcasOBP9A and TcasOBP9B from T. castaneum beetle as sensing elements. These sensor devices respond to different concentration of 3-octanol and 6-methyl-5-hepten-2-one, even at a low concentration in real-time affinity assays. Based on this results, in comparision to portable biosensor available in the market, our new rGO-FET biosensor improved detection limit and sensitivity of odor molecules and can be used for directly measuring the affinities of interested ligand with binding protein. The obtained results from artificial sensor device is compared with an EAG response of the whole antenna of a living beetle by using fitting data based on the Langmuir model. This study represents a first step towards the development of olfactory sensor device closely mimicking the complex natural olfactory system of an insect antenna.

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