Risultati finali
ORs belong to the largest and important class of GPCR receptors (G protein-coupled receptors), sharing with this kind of receptors the seven-helices transmembrane structure. Unfortunately, the information about the structure at atomic level is missing for ORs while it is available for rhodopsin (GPCR photonic receptor). Therefore, we have developed a model, which takes advantage of the common coarse grain structure of ORs and bovine rhodopsin.
Our device under test (DUT in the following) is a single molecule of bovine rhodopsin, inserted between two metallic contacts in some environment (membrane, biological solution, antibodies, etc.).
The DUT is modelled as an equivalent circuit consisting of an impedance network (IN). The equivalent circuit can be presented as a simple non-directed graph. The nodes (vertices) of this graph correspond to the aminoacids (aminoacid residues) of the protein (348 for the bovine rhodopsin) and the links (edges) between any couple of nodes characterise some kind of interaction between aminoacids, which are neighbouring in space within a given radius. Two more nodes acting as contacts are then introduced.
The contacts are linked to a given set of aminoacids (each contact linked to at least one aminoacid). The environment is not taken into account at this stage. The elementary impedance (impedance of the link) is taken as the most usual passive equivalent AC circuit made of a resistor in parallel with a capacitor.
To construct the IN, the aminoacids are considered as uniform spheres with the centre in the alpha carbon atom and a typical van der Waals radius, Ra=5Å. When two spheres overlap, they are assumed to be neighbouring and the impedance edge should be inserted between the corresponding nodes of the circuit. Otherwise there is no interaction between aminoacids and thus no link between the corresponding nodes.
The determination of the value of each impedance can be fixed with increasing degree of complexity. Here we consider three different approaches.
(i) As first approach, the impedances of the edges are taken to be all the same.
(ii) In a second approach, Zi,j is taken to be proportional to the li,j as for a simple ohmic resistor and planar homogeneous capacitor.
(iii) In a third approach, by assuming that the cross-sectional area of resistor and capacitor is equal to the cross-section area of overlapping spheres.
Further approaches can be introduced to allow including properties of real aminoacids. In going from model (i) to (iii) we exploited a better sensitivity of the network to the change of its structure. For the three models presented it can be observed a systematic decrease of |Z| at increasing Ra (radius of electrical interaction) reflecting the increasing importance of parallel with respect to series connections. The continuous increase of Ra is equivalent to a continuous three-dimensional contraction of the structure and represents an example of a spatial conformation dependence of |Z|. Everywhere below, by default we use the model (iii).
Such an equivalent circuit can be used for modelling the network with the value of all the elementary impedances fixed (perfect impedance network) or with each elementary impedance allowed to take two or more values stochastically thus becoming a Random Impedance Network.
For the comparison of two spatial configurations of the same molecule one should build equivalent circuits for both of them. Even if the primary structure of the molecule remains the same, distances between aminoacids li,j change and therefore the topology of the network will in general change. In the second configuration some new links should arise, some old should disappear. In particular, when the elementary impedances depend on distances, the circuit should become even more sensitive to the change of configuration.
The main conclusion that can be drawn is that the maximum variation of impedance due to a conformational change is expected up to about 30% and corresponds to a an interacting radius in the range 5 < R < 25 A
This prediction concerns a frozen network (static network) in which the atomic positions are fixed at the equilibrium values. On the other hand, fluctuations of the atomic positions, due to thermal motion imply an impedance noise, whose level, in comparison with the impedance variation due to the conformational transition and with the electrode/amplifier noise, is crucial to the actual detection of the ligand capture by the GPCR. Therefore, here we extended the previous model to include the effect of the thermal motion on the electrical response to an AC field. To this purpose, we applied a classical harmonic oscillator and then consider the case of a quantum harmonic oscillator.
Apart from interesting properties on the statistical properties of the impedance network we have shown that the thermal random fluctuations will not mask the expected electrical response upon the conformational change.
Prototypes of Atomic Force Microscope probes for electrical measurements in liquid media have been designed and fabricated.
The structure of the probes is a cantilever in the plane surface of a silicon chip with a tip in the upward direction at its end. They include a conducting layer for electrical measurements. The cantilevers are fabricated on silicon nitride with the tip made of polycrystalline silicon. The conducting layer is gold for biocompatibility reasons.
In the fabrication process the following aspects have been implemented:
- The backside photolithography is made at the beginning, before the fabrication of the tip, to avoid damaging the tips.
- A metallisation scheme conssting of 2 nm Cr to improve the adhesion of gold plus 20nm Au has been used, to avoid etching problems with the initial one (Ti/Ni/Au);
-The thicknesses of the silicon nitride layers has been corrected to improve the residual stress behaviour of the cantilever. The new values are 250nm for the structural layer and 200 to 300nm for the passivation layer;
- An innovative photolithography method to eliminate the passivation dielectric layer only at the apex of the tip, thus avoiding parasitic parallel current paths near the cantilever tip when measuring in an aqueous medium. The method is based on depositing a thick photoresists that covers the chip but not the tip apex.
The structure of the chips containing the cantilevers is defined by wet silicon micromachining, to avoid the silicon wafer sawing process that would damage the fragile cantilevers and tips. Chips have been designed with both rectangular and V-shaped cantilevers, with spring constants ranging from 0.01N/m to 1N/m, suitable for contact mode scanning of biological samples. Cantilever lengths range from 150 to 300m and widths from 30 to 60um.
ORs belong to the largest and important class of GPCR receptors (G protein-coupled receptors), sharing with this kind of receptors the seven-helices transmembrane structure. Unfortunately, the information about the structure at atomic level is missing for ORs while it is available for rhodopsin (GPCR photonic receptor). Therefore, we have developed a model, which takes advantage of the common coarse grain structure of ORs and bovine rhodopsin.
Our device under test (DUT in the following) is a single molecule of bovine rhodopsin, inserted between two metallic contacts in some environment (membrane, biological solution, antibodies, etc.).
The DUT is modelled as an equivalent circuit consisting of an impedance network (IN). The equivalent circuit can be presented as a simple non-directed graph. The nodes (vertices) of this graph correspond to the aminoacids (aminoacid residues) of the protein (348 for the bovine rhodopsin) and the links (edges) between any couple of nodes characterise some kind of interaction between aminoacids, which are neighbouring in space within a given radius. Two more nodes acting as contacts are then introduced.
The contacts are linked to a given set of aminoacids (each contact linked to at least one aminoacid). The environment is not taken into account at this stage. The elementary impedance (impedance of the link) is taken as the most usual passive equivalent AC circuit made of a resistor in parallel with a capacitor.
To construct the Impedance Network, the aminoacids are considered as uniform spheres with the center in the alpha carbon atom and a typical van der Waals radius, Ra=5Å. When two spheres overlap they are assumed to be neighbouring and the impedance edge should be inserted between the corresponding nodes of the circuit. Otherwise there is no interaction between aminoacids and thus no link between the corresponding nodes.
The determination of the value of each impedance can be fixed with increasing degree of complexity. Here we consider three different approaches.
(i) As first approach, the impedances of the edges are taken to be all the same.
(ii) In a second approach, Zi,j is taken to be proportional to the li,j as for a simple ohmic resistor and planar homogeneous capacitor.
(iii) In a third approach, by assuming that the cross-sectional area of resistor and capacitor is equal to the cross-section area of overlapping spheres.
Further approaches can be introduced to allow including properties of real aminoacids.
In going from model (i) to (iii) we exploited a better sensitivity of the network to the change of its structure. For the three models presented it can be observed a systematic decrease of |Z| at increasing Ra (radius of electrical interaction) reflecting the increasing importance of parallel with respect to series connections. The continuous increase of Ra is equivalent to a continuous three-dimensional contraction of the structure and represents an example of a spatial conformation dependence of |Z|. Everywhere below, by default we use the model (iii).
Such an equivalent circuit can be used for modelling the network with the value of all the elementary impedances fixed (perfect impedance network) or with each elementary impedance allowed to take two or more values stochastically thus becoming a Random Impedance Network.
For the comparison of two spatial configurations of the same molecule one should build equivalent circuits for both of them. Even if the primary structure of the molecule remains the same, distances between aminoacids change and therefore the topology of the network will in general change. In the second configuration some new links should arise, some old should disappear. In particular, when the elementary impedances depend on distances, the circuit should become even more sensitive to the change of configuration.
The main conclusion that can be drawn is that the maximum variation of impedance due to a conformational change is expected up to about 30% and corresponds to a an interacting radius in the range 5 < R < 25Å
This prediction concerns a frozen network (static network) in which the atomic positions are fixed at the equilibrium values. On the other hand, fluctuations of the atomic positions, due to thermal motion imply an impedance noise, whose level, in comparison with the impedance variation due to the conformational transition and with the electrode/amplifier noise, is crucial to the actual detection of the ligand capture by the GPCR. Therefore, here we extended the previous model to include the effect of the thermal motion on the electrical response to an AC field. To this purpose, we applied a classical harmonic oscillator and then consider the case of a quantum harmonic oscillator.
We have shown that the thermal random fluctuations will not mask the expected electrical response upon the conformational change.
The membrane fraction obtained from the yeast cells expressing the ORs consists, among other things, of nanosomes whose size can be controlled.
In the initial plan of the project it was foreseen the possibility to purify the receptors from the yeast cell in order to further immobilize them on the substrates by using techniques similar to the Langmuir-Blodgett technique. However, two main results obtained inside the projected suggested that other strategies should be addressed. On the one hand it was verified that the expression level even if high it is not high enough to be subject to a standard purification process (comparable to the purification process followed inside the project for bovine rhodopsine in the first stages of the project). On the other hand, even if purification would have been possible, it was demonstrated that the formation of reconstituted films of membrane proteins on solid substrates by the Langmuir-Blodgett technique was not possible, as verified with purified samples of bovine rhodopsin inside the project.
These results suggested an alternate immobilization strategy. The one that finally was implemented implied an in-situ purification of the produced membrane fraction on previously functionalized substrates.
To this end, it was necessary to perform a thoroughly characterisation of the membrane fraction obtained from the lyses of the yeast cells expressing the OR I7.
Characterization was performed by Transmission Electron Microscopy and Atomic Force Microscopy.
The membrane fraction was prepared as detailed: Yeast cells resuspended in ice-cold lysis buffer (50mM Tris-HCl, pH 7.5, 1mM EDTA, 0.1mM PMSF, 250mM sorbitol) and the complete protease inhibitor cocktail can be disrupted by either of 2 methods:
- Vortexing with glass beads. Glass beads are added and cells are disrupted by 7 cycles of 1 min of vigorous vortexing/1 min of cooling on ice.
- Cell disintegration using a Cell Disrupter "Basic Z" (Constant System Ltd). Yeast cells can be disrupted at 4°C at a pressure of 1200 bars.
In both cases, samples are then centrifuged at 5000g for 10 min at 4°C to remove unbroken cells and cell walls. The supernatant is further centrifuged at 40,000 g for 40min at 4°C. This second pellet, enriched in plasma membranes, is resuspended in the lysis buffer with a Dounce homogeniser, and stored in aliquots at 80°C.
Negative staining electron microscopy of this membrane fraction was performed.
It is thus clear that the fragments are indeed circularised into microsomes: the bilayer is visualised at the outline of the structures. Their size ranges from hundreds of nm (large structures, microsomes) to tens of nm (small structures, nanosomes).
A further sonication of the samples was used to reduce the size of the fragments present in these membrane fractions and make it uniform (sonication bath). A number of experimental parameters should be considered: power output level, duty cycle, total sonication time.
An additional 20 minutes sonication indeed yields nanosomes of uniform size (40-60nm).
From these studies we concluded that the preparation procedures used provide membrane fraction samples mainly containing nanosomes of uniform (40-60nm) size. This size seems to be in good adaptation with the geometrical requirements of the other workpackages of the project.
Concerning Atomic Force Microscopy characterization structural characterisation of the nanosomes containing the olfactory receptors provided by WP1 once immobilised on a solid substrate and in liquid environment has been performed with the AFM set up. Experiments have been performed on both bare and functionalised gold substrates in order to check the effects of the hydrophobicity on the structural properties of the nanosomes.
The aspect ratio of the nanosomes once adsorbed is quite low (typically below 1:3). This rule is followed by a wide range of measured nanosome sizes and for the two types of surfaces analysed.
We note that effects of the hydrophobicity are not evident from the measurements performed.
Finally, we analysed the surface coverage of the adsorbed nanosomes. While on non sonicated samples the coverage is quite poor (below 10%), after sonication coverages close to the 50% have been measured on bare gold. In addition to higher surface coverage, homogenisation of the sizes around 50 nm of the nanosomes has been noticed.
Finally, we have performed Transmission Electron Microscopic images of single nanosomes to provide a more direct prove of the presence of olfactory receptors in them. Some protuberances of size comparable to a single olfactory receptor are evident on the nanosome surface, although not all of them would correspond to olfactory receptors. Some of the images obtained show a single nanosome containing OR 1740 preparaded by inmunostaining with gold nanoparticles of 5nm diameter, demonstrating that some ORs are present in the nanosome.
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.
There exist in the market available electronic instrumentation for electrochemical characterisation of a wide variety of samples. Among them we cite the equipments used inside the present project for the characterisation of the microtransducers: The Voltalab system sensibility is intended for measurements at the milimeter scale, while brand new systems are intended for more sensitive measurements and hence adequate for measurements at the micro/nanoscale. However, these last systems even if powerful are very expensive and not very versatile hence not suitable for the development of a practical biosensor
For these reasons, we have developed a full custom electrochemical electronic instrumentation for the measurements with the micro/nanotransducers developed inside the project.
The measuring system consists on an electronic front-end setting and reading the signals in the Control Electrode, in the Working Electrode and in the Reference Electrode, featuring a special circuitry for spurious current compensation due to interelectrode capacitance of any value.
Two electronic front-ends have been fabricated in order to adapt the instrumentation both for microelectrodes and nanoelectrodes. The front-end for the microelectrodes can draw a maximum DC current of 1µA with a signal bandwidth of 2MHz. For the nanoelectrodes a second front-end has been fabricated with ten times less instrumental noise increasing proportionally the sensitivity. The disadvantage of this solution is a reduced maximum DC current (10nA) and signal bandwidth (0.9MHz).
The electronic front-ends are miniaturised to be lodged close to the electrochemical cell, and are followed by the control electronic stage providing more amplification, the power supply and the electronic interface to connect with a PC for automatic control.
The connection to the nanoelectrodes has been optimised with a proper board that allows an easy fit of the nanochip into the electrochemical cell.
The fabricated instrument is controlled by a PC with a data acquisition board and can perform both cyclic voltammetry (resolution of 40fA at the maximum speed of 1V/s) and impedance spectroscopy (signal bandwidth 1Hz 0.9MHz, tens of attoFarad of capacitance resolution).
Software to control the instrumentation for electrochemical measurement has also been realised.
The main features are:
- Generation and acquisition of the signals for cyclic voltammetry; all parameters for the measurement (voltage curve, speed, number of cycles) are easily selectable;
- Visualisation of the voltammetry measurements with different filters to reduce spurious noise;
- Generation and acquisition of the signals for electrochemical impedance spectroscopy; all parameters for the measurement (dc bias, ac amplitude, frequency range, averaging time) are easily selectable;
- Visualisation of the impedance measurements as a Cole-Cole plot (real part vs imaginary part of the impedance) or as a Bode plot (impedance module and phase as a function of the frequency);
- Automatic calibration of the instrument to compensate phase shift due to the cables and the PoliMi amplifier;
- Tracking the time evolution of the impedance at a single frequency;
- All the measurements are available in standard ASCII format.
With the Atomic Force Microscope set up developed in the project we have developed a method to characterize the electrical properties of biological samples with nanoscale spatial resolution. The method implemented uses dynamic or jumping mode to image the sample topography and then contact mode approach to perform the electrical measurements.
We have performed a number of DC and AC measurements on a number of biological samples, and in particular on a single nanosomes containing olfactory receptors.
We have verified the following properties:
- The nanosome behaves as an insulator with respect to DC electronic transport, with a resistance far above hundreds of GigaOhms for the AFM tip-nanosome-gold substrate system. This result has been further confirmed with DC measurements performed on bacteriorhodopsin purple membrane only 5 nm thick. In this system only direct tunnel electric current has been observed with an AFM tip-purple membrane-gold substrate resistance of hundreds of GigaOhm.
- The nanosome displays an almost pure capacitive behaviour for AC electronic transport between 100Hz up to 300kHz. In the nanosomes studied no significant frequency dispersion on the electrical properties has been observed. However, on bigger nanosomes this result may need further investigation, since the water enclosed inside the nanosome could produce frequency dispersion phenomena.
-The relative dielectric constant of the whole nanosome has been estimated to be around 2. This value has been extracted from a capacitance distance curve performed on a single nanosome and comparing it with a capacitance distance curve performed on the gold substrate and with the help of a theoretical model involving realistic formulas for the capacitance of the AFM probe-substrate system.
The fact that the measurement has been performed on a single nanosome can be verified by a post measurement imaging of the nanosome, where the tip indentation can be clearly identified.
Finally, some attempts to perform an electrical map of the electrical properties of nanosomes were essayed in contact mode by using a softer cantilever (0.2 N/m). Even though capacitance images were efficiently recorded by the measuring set up and results are in qualitative agreement with capacitance-distance curve measurements, some concerns on the sample integrity remained after the images were performed.
We have integrated all the developments carried out during the project in an olfactory biosensor prototype consisting of:
- A microtransducer consisting of a microelectrochemical cell.
- A single layer of nanosomes incorporating the selected olfactory receptor and immobilised through the mixed functionalisation technique developed.
- An electrochemical nanoscale amplification electronic circuit.
- An acquisition board plus a user-friendly interface for the data acquisition and processing.
- A liquid cell specifically designed for the nanobiosensor with liquid injection pumps for odorant injection.
In any case, even if not fully demonstrated, the developed set up theoretically satisfies the requeriments it should meet to work as a biosensor based on the electrical properties of olfactory receptors with sensibility down to a single nanosome, that was one of the main objectives of the project.
Prototypes of micro/nanotransducers based on a scaling down of an electrochemical impedance spectroscopy cell have been developed.
The chips have been designed with a long rectangular shape (13.0 x 1.0mm{2}), to facilitate the electrical isolation of the sensing part, to be immersed in a liquid solution, from the electrical connection pads. Much of the chip area consists on micron-sized conducting elements and electrodes.
Various designs of microelectrodes have been produced, with either square or circular shapes. The circular electrodes have been designed with the following diameters: 80, 60, 40, 20, 15, 10, 5um and 500nm. This allows a scaling down of the electrochemical impedance spectroscopy measurements.
The microelectrode devices are fabricated on wafer level by using standard microelectronic processes. To obtain nanoelectrodes (500nm), the gold electrodes are left covered with a silicon dioxide layer. This is later opened by a nanolithography process. This can be done by electron beam lithography at a wafer level (a serial process but with 24 chips at a time), followed by oxide etching, or directly by focused ion beam etching each chip. Both individual nanoelectrodes and nanoelectrode arrays have been fabricated. Various final diameters from about 1 micron to less than 500 nm have been successfully obtained.
The platinum layer is used to define counter-electrodes and also reference microelectrodes on chip. To obtain a reference electrode, an additional layer of silver is deposited on top of one of the platinum electrodes. The silver is then electrochemically chlorinated in an HCl solution to obtain an Ag/AgCl reference electrode.
In relation to the chip packaging the requirement of usage in a liquid medium adds some constraints to the packaging design. The wires making electrical contact between the chip and the external system must be protected, but the micro/nano-electrode area should be in contact with the liquid. For this reason the micro- and nano-electrode chips have been defined with a long geometry, to separate the liquid from the electrically active area. The chips have been bonded to specific printed circuit boards (PCB), wire-bonded and covered with a polymer to protect the chip. The wire bonding is further protected by a polymeric (epoxy) cover, which is applied using a mould.
With the objective of characterizing the electrical properties (both DC and AC) with nanoscale spatial resolution a full custom electronic instrumentation implemented on a commercial Atomic Force Microscopy has been developed.
The devloped set-up consists of a commercial Atomic Force Microscope (Nanotec Electronica, S.L.) coupled to a custom-made transimpedance amplifier that has been implemented to allow a multifunctional electrical characterisation with AFM. It is based on the integrator-differentiator scheme, but provided with an additional feedback loop to discharge the dc current. The amplifier can measure ac current over a wide spectrum, ranging from few Hz to MHz, simultaneously to dc current measurement, with a flat noise density of around 10fA/vHz up to10kHz and no time constraints. Thus, the implemented amplifier proves to be suited for a multipurpose electrical characterisation using AFM, namely IV measurement, impedance and noise.
The present set up for DC/AC electrical characterization at the nanoscale is not currently available in any commercial Atomic Force Microscope.
Its field of applications range from the characterization of thin film oxides in Electronics to self-assembled monolayers or biomembranes and liposomes in biochemistry and biosensor development.
The effect of specific odorant (heptanal and octanal) and non-specific odorant (helional) on the electrical properties of the biofilm formed by the self-assembled monolayer, the antibody and the membrane fraction including the olfactory receptor has been shown by electrochemical impedance spectroscopy. Despite of non-specific effects for concentration of odorant higher than 10E-9M, specific recognition appears clearly with concentrations of odorant between 10E-13M and 10E-9M.
Many test controls were performed with self-assembled monolayer including the single antibody, including the specific antibody and rhodopsin and the specific antibody and olfactory receptor ORI7. The effect of solvent DMSO is less than 10%.
In such tests it clearly appears a higher specificity of ORI7 for heptanal and for octanal.
Cmyc-OR 1740 was anchored on the mixed SAM using the monoclonal anti-cmyc antibody. The specificity of OR 1740 for helional, compared to heptanal has been studied. An optimum sensitivity is observed for a concentration of 10-10M of helional. It has been checked that without any OR, the mixed SAM presents no variation of polarisation resistance in presence of helional, whatever its concentration.
The effect of the presence of GTP on the sensitivity of detection has been studied by the same technique. The presence of GTP increases the sensitivity of detection by a factor of 6 due to an amplification effect already observed by SPR measurements.
This result constitute one of the main results of the project and prove one of the hyphothesys on which it was based, namely, that biosensors based on the electrical response of olfactory receptors can be developed.
We used Surface Plasmon Resonance technique for monitoring the functional response of the olfactory receptors in the nanosomes once immobilized on solid suports.
The procedure consisted on two steps: in the first step immobilization of the nanosomes on the sensorchip was verified while in the second specific response of the immobilized nanosomes to odorants were monitorized. Previuous test experiments with bovine rhodopsine membrane fraction were performed.
For the verification of the nanosome immobilization the membrane fraction corresponding to yeast cells expressing rat I7 was injected on top of a L1 sensor chip (gold surface covered with dextran modified with lipophilic compounds). Then subsequently the polyclonal anti-I7 antibody was injected. No detection could be obtained, even for highly concentrated membrane fraction deposition. One possible explanation is that the specific IgG is not concentrated enough within the anti-I7 polyclonal antibody.
Even though no response was obtained in this case, the functional response tests proceeded. The straightforward idea would be to inject the odorant solution on the BIAcore sensor chip after immobilisation of the membrane fraction containing the olfactory receptor. However, since the odorants used are so small, their binding to the receptors of the membrane fraction cannot be directly detected. We thus decided to use a procedure set up by Vogel (Bieri et al.) to follow activation of rhodopsin.
Golf departure is indeed detected upon heptanal (5mM) stimulation of the I7 receptor present in the membrane fraction deposited on the L1 sensor chip (0.004mg/ml total protein concentration in membrane fraction deposited), in the presence of 10mM GTP.
In summary, the olfactory receptor is indeed still in its active form in the membrane fraction deposited onto the sensor chip. Golf is still present in the membrane fraction, pre-associated with the olfactory receptor. Stimulation of the olfactory receptor by its odorant ligand induces its conformational change, and thus its interaction with the a subunit (Golf) of the G protein, which dissociates from the trimer. This event is detected by the BIAcore.
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