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Electrochemical study of human olfactory receptor OR 17–40 stimulation by odorants in solution
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Materials Science and Engineering C 28 (2008) 633 – 639 www.elsevier.com/locate/msec
Electrochemical study of human olfactory receptor OR 17–40 stimulation by odorants in solution I.V. Benilova a,c,⁎, J. Minic Vidic b , E. Pajot-Augy b , A.P. Soldatkin c , C. Martelet a , N. Jaffrezic-Renault d,1 b
a Laboratoire AMPERE, UMR-CNRS 5005, Ecole Centrale de Lyon, 69134 Ecully Cedex, France INRA, Neurobiologie de l'Olfaction et de la Prise Alimentaire, Récepteurs et Communication Chimique, Domaine de Vilvert, 78352 Jouy-en-Josas Cedex, France c Laboratory of Biomolecular Electronics, Institute of Molecular Biology and Genetics NASU, 03143 Kyiv, Ukraine d Université Claude Bernard—Lyon 1, Laboratoire des Sciences Analytiques, UMR-CNRS 5180, 69622 Villeurbanne Cedex, France
Available online 13 October 2007
Abstract The human olfactory receptor OR 17–40 co-expressed with α-subunit of Golf protein in yeast was attempted as a bio recognition part of impedimetric biosensor. The receptor in its natural membrane environment was anchored to a gold-coated glass substrate modified with thiolbased multilayer. Step-by-step building-up of the OR 17–40 based biofilm was monitored using surface plasmon resonance technique. Stimulation of the OR 17–40 with its cognate odorant helional in phosphate-buffered saline was probed by means of electrochemical impedance spectroscopy under various conditions. Activation of OR 17–40 in the presence of GTP-γ-S at 4 °C was found to improve the sensitivity of the developed labelfree biosensor, probably via the enhancement of the specific biochemical signal. © 2008 Elsevier B.V. All rights reserved. Keywords: Impedance spectroscopy; SPR; Bioelectronic nose; Human olfactory receptor; Helional
1. Introduction Simultaneous detection of different odorants in liquid and gaseous mixtures is extremely important for quality control in food, beverage and fragrance industries. Nowadays, the instruments capable of a broad-band routine analysis of complex aroma are mainly represented by mass spectrometers, or gas chromatographs combined to mass spectrometers (GC/MS). However, a spectrum of odor peaks obtained by means of GC/MS does not actually provide sufficient information about aroma quality itself which, however, directly arises from the human odor impressions [1]. The arrays of semiselective sensors coupled to the patternrecognition systems and called artificial (electronic) noses [1–3] ⁎ Corresponding author. Université Claude Bernard—Lyon 1, bât. Jules Raulin, 5ème étage, Laboratoire des Sciences Analytiques, UMR-CNRS 5180, 69622 Villeurbanne Cedex, France. Tel.: +33 4 72 43 11 82; fax: +33 4 72 43 12 06. E-mail address: [email protected] (I.V. Benilova). 1 Université Claude Bernard—Lyon 1, bât. Jules Raulin, 5ème étage, Laboratoire des Sciences Analytiques, UMR-CNRS 5180, 69622 Villeurbanne Cedex, France. 0928-4931/$ - see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2007.10.040
present another kind of analytical instrumentation that mimics natural olfactory system operation. As sensitive elements of electronic noses, conductive organic polymers [4,5], porphyrins [6], calixarenes [7–9], can be used. In the last years, the curiosity of scientists to achieve a deeper understanding of the functional peculiarities of olfactory system of vertebrates has stimulated the development of a bioelectronic nose [1,10]—multisensor device whose biorecognition part consists in natural G protein coupled olfactory receptors. Many recent reports concern the elaboration of individual sensors based on the ORs expressed either in olfactory sensory neurons [10] or in heterologous cells [11–14] and coupled to various transducers like quartz-crystal microbalance [13,14], lightaddressable potentiometric sensor [10], substrates for the SPR (surface plasmon resonance) [11] and the EIS (electrochemical impedance spectrometry) [12] techniques. Some authors consider the human olfactory receptors to be narrowly selective for the individual odorants in contrast to the rodent ORs which are able to respond to a large repertoire of ligands [15]. However, the sensitivity of human nose does not
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arise from the values of binding constants of odorant-receptor coupling itself but rather from the signal processing in the higher brain structures [2], and therefore a choice of appropriate data processing model is an extremely important step in the bioelectronic nose elaboration. Artificial neural networks are one of the most widely used pattern-recognition models [16,17] dealing with complex nonlinear dose-dependence relations and therefore can be very suitable for the bioelectronic noses. However, dealing with time-consuming complex training of such networks requires very high stability of ORs coupled to the transducers and a satisfactory low level of stochastic responses as well. Previously, our group has studied the sensitivity of heterologously expressed rat OR I7 [12] and human OR 17–40 [11] towards a set of different odorants, by means of the EIS and the SPR technique, respectively. In this work, for the first time, a stimulation of SPR chip-coupled OR 17–40 by two ligands, preferential (helional) and unrelated (heptanal), was probed by means of nonfaradaic EIS. In order to improve a resolution of such an impedimetric label-free sensor via the enhancement of the biochemical signal, ligand-receptor interactions were also investigated in the presence of the guanosine triphosphate (GTP-γ-S). 2. Materials and methods 2.1. Biomaterials and chemicals Gαolf protein and human OR 17–40 tagged with cmyc sequence on its N-terminus were co-expressed in yeast Saccharomyces cerevisiae (strain MC18) [18] and the membrane fraction was prepared as described in [11]. Stock suspension of membrane fraction with protein content 2.85 mg/ml was aliquoted and frozen at − 80 °C. Monoclonal anti-cmyc antibody (Ab) was obtained from Roche Molecular Biochemical and biotinylated by means of DSB-X™ Biotin Protein Labeling Kit (Molecular Probes, Leiden, Netherlands). Stock solution of Ab (2.55 mg/ml) was divided into aliquots and stored at −20 °C. 16-Mercaptohexadecanoic acid (90% purity) and 1,2dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-biotinyl sodium salt (biotinyl-PEA) were purchased, respectively, from Aldrich and Avanti Polar Lipids. GTP-γ-S (guanosine-5′-O-(3thiotriphosphate); MW 563, 93% purity), bovine serum albumin (BSA; 98% purity) and goat IgG were obtained from Sigma, neutravidin—from Pierce. Ethanol (99.8%), HCl (37%), HNO3 (65%), H2O2 (30%) and NH4OH (25%) were purchased from Fluka and used as received. Dimethyl sulfoxide (DMSO) for odorant dilution and heptanal were obtained from Sigma. Helional was a kind gift from Givaudan-Roure (Switzerland). As a working buffer, a phosphate-buffered saline (PBS) was used with the following composition: 8 mM Na2HPO4, 1.5 mM KH2PO4, 3 mM KCl, 150 mM NaCl, pH 7.0 [19]. All reagents for PBS preparation were of analytical grade. All aqueous solutions were prepared with ultrapure water from Milli-Q system.
2.2. SPR technique and gold-coated substrates Surface plasmon resonance spectrometer “Biosuplar 3” (www.micro-systems.de) was developed at the Institute of Semiconductor Physics of the National Academy of Sciences of Ukraine (Kyiv). This optoelectronic device based on the phenomenon of the surface plasmon resonance in the Kretchmann's optical configuration was controlled by a computer via self-developed software. Gold film (45 nm) deposited through Cr adhesion layer (1–1.5 nm) onto a glass chip represented the sensor surface. An incident beam of ppolarized light from a semiconductor laser diode (λ = 650 nm) excited an oscillation of electronic plasma (i.e. surface plasmon) in this metallic film. A special prism capable of rotation on a computer-defined angle provided optimal conditions for the plasmon excitation. A plasmon resonance itself was registered as a drastic decrease of reflected light intensity. In the present study, the SPR spectrometer flow cell (V∼20 μl) was connected to a Gilson Minipuls 3 pump. 2.3. Pretreatment of sensor surface Before work, substrates were cleaned by a mixture “aqua regia” (H2O + HCl + HNO3, 16 + 3 + 1, v/v) during 1–1.5 min and then—with a basic mixture (H2O + H2O2 + NH4OH, 5 + 1 + 1, v/v) during 15 s. Thoroughly rinsed with water, every chip was then immersed into ethanol for several seconds. 2.4. Self-assembly of the mixed layer onto gold To obtain mixed self-assembled monolayer (SAM) on gold surface, 1 mM mercaptohexadecanoic acid and 0.1 mM biotinyl-PEA dissolved in ethanol were incubated with freshly cleaned chip during 21 h [20]. Mercaptohexadecanoic acid is fixed onto Au via the chemisorption of SH-groups, as to biotinyl-PEA, it can be inserted between long-chain thiols due to the numerous hydrophobic interactions. Such mixed SAM provides a good basis for the further anchoring of biomolecules to the chip surface. To elute unfixed molecules, chip was rinsed with ethanol, and dried under a nitrogen flow. 2.5. Blocking step and formation of the upper supporting layers In order to saturate all non-specific adsorption sites on SAM surface, heterogeneous layer was treated by 1 mM solution either of IgG or BSA in PBS in the flow cell: 0.3 ml of blocking molecules solution was run on the chip at the flow rate (FR) 0.04 ml/min. Neutravidin (0.5 μM, 0.3 ml solution in PBS) and biotinylated anti-cmyc Ab (0.5 μM, 0.3 ml solution in PBS) were subsequently run on the blocked SAM at the FR 0.04 ml/min. Before the formation of any upper molecular layer, the previous one was rinsed with PBS during 5–15 min. 2.6. Preparation and immobilization of OR 17–40 Stock suspension of OR 17–40 in its natural membrane fraction was thawed and resuspended in PBS on ice up to the
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OR concentration 70 μg/ml. 0.3 ml of this suspension was treated in the ultrasonic bath Elmasonic (35 kHz, 120 W) in icecold water during 10 or 20 min in order to obtain more or less heterogeneous suspension of membrane nanovesicles [11]. Afterwards, 0.3 ml of this suspension was immediately run on the chip (FR 0.04 ml/min). 2.7. Electrochemical probing After immobilization of the OR 17–40, SPR chip was washed with PBS and transferred into a three-electrode electrochemical glass cell (V = 5 ml) placed into a Faradaic cage and filled with PBS. The EIS measurements were performed with an impedance spectrometer Voltalab 40. In order to conduct measurements at 4 ± 0.5 °C, the cell was connected to the circulator thermostat Julabo F25 (France). All other measurements were performed at room temperature (20 ± 1 °C). As a reference a saturated calomel electrode (SCE) from Radiometer Analytical was employed, and a platinum plate was used as an auxiliary electrode. Active surface area of the latter was 0.64 cm2, and 0.25 cm2 for the working electrode (i.e. biofunctionalized chip). Impedance was swept in the frequency range 105–0.1 Hz at dc potential − 700 mV vs SCE with superimposed excitation voltage of 10 mV. Data were visualized as Cole–Cole (Nyquist) plots via the VoltaMaster 4.0 software. 2.8. Preparation of odorant solutions Only freshly prepared solutions of helional and heptanal were used in all experiments. Stock 0.1 M solutions were prepared by the dilution of corresponding aldehyde in DMSO, but further dilutions were performed only in PBS. 10− 3 M dilution was directly prepared from 0.1 M, then further dilutions were obtained by successive 1:10 dilutions. To take into account a possible solvent effect in biosensor response, blank probes at the various dilutions were prepared, replacing the helional by PBS. 2.9. Monte-Carlo simulation In order to estimate the impact of nanosomes size on their efficient immobilization, a Monte-Carlo simulation was used. A correspondent model was created using the MATLAB 7.0 software. 3. Results and discussion 3.1. Layer-by-layer assembly of OR-containing biofilm A typical kinetics of step-by-step formation of the ORcontaining biofilm is shown in Fig. 1A. At first, PBS was run on the chip surface functionalized with SAM; afterwards it was treated with IgG or BSA. Adsorption of BSA onto the surface produced a signal shift of about 0.12 arc degrees (Fig. 1A), while the response to the IgG anchoring was about 2.5 times higher (data not shown), probably due to the difference in the molecular mass of BSA (66 kDa) and IgG (150 kDa).
Fig. 1. (A) Typical building-up kinetics of the OR-containing SAM-based biofilm. (B) SPR responses to the step-by-step formation of OR-containing multilayer. All measurements were performed in the flow cell in PBS at pH 7.0.
The next step of surface modification consisted in the immobilization of neutravidin. Neutravidin (60 kDa) presents an interesting alternative to the streptavidin since it is a neutral protein without any carbohydrate moieties, thus possessing rather low capabilities of non-specific binding at physiological pH [21]. Neutravidin molecule has 4 binding sites (two on each opposite sides), presenting a very suitable linker for the consecutive anchoring of any biotinylated molecules. As it can be seen from Fig. 1B, anchoring of neutravidin to the BSAblocked or IgG-blocked SAM produces a resonance angle shift of about 0.2–0.25 arc degrees. However, coupling of neutravidin to the non-blocked SAM produced a higher signal presumably due to some spatial restriction of accessibility of biotin groups after BSA/IgG blockage step. Biotinylated anti-cmyc Ab was immobilized over the neutravidin layer. Anchoring of Ab onto the surface blocked either with IgG or BSA produced a signal of about 0.3 arc degrees being 1.7 times weaker than it could be expected from the molecular mass ratio Ab : neutravidin = 2.5. Maximal SPR response to Ab anchoring (0.55 arc degrees) was achieved on the non-blocked neutravidin layer, however, the average binding
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capabilities of the latter were shown to be statistically the same as those of the BSA and IgG-blocked structures (Fig. 1B). Finally, a suspension of OR 17–40-containing nanosomes was run on the surface and incubated for about 20–30 min, then rinsed with PBS. The best average SPR signal for the OR 17–40 immobilization was obtained on the basis of the BSA-blocked structure (Fig. 1B). The latter was even more efficient than the non-blocked structure demonstrating a good accessibility and a proper orientation of the anti-cmyc binding sites of Ab. As to the IgG-blocked multilayer, it was 3 times less capable of nanosomes binding than the biofilm blocked with BSA. The impact of ultrasonication duration on the nanosomes adsorption on the BSA-blocked surfaces was also investigated. It was found that nanovesicles pretreated during 10 min produced the SPR signal of 0.26 ± 0.07 arc degrees (n = 8), while those sonicated during 20 min yielded 0.22 ± 0.03 arc degrees (n = 5). So, the SPR chips modified by the BSA-blocked multilayer coated with OR-containing nanosomes sonicated during 10 min were chosen for further electrochemical studies. A step-by-step formation of OR-containing biofilm took about 280–330 min and was a fully real time controllable process. 3.2. Impedance probing of SPR sensor chips After the OR-containing biofilm formation, the SPR chip was employed as the working electrode in the electrochemical cell. Multilayer revealed a high stability upon contact with PBS solution and rather high complex impedance as well (Fig. 2). As a working potential, − 700 mV vs SCE was chosen and applied for all measurements. Under this potential the OR-containing biofilm remained stable in time, and crude complex impedance data (e.g., like in Figs. 3 (A,B) and 5 (A,B)) gave a good fit to an electrical circuit shown in Fig. 4 over the broad frequency range. The above-mentioned circuit taken for data processing is composed of the ohmic resistance of bulk electrolyte (Rs) in
Fig. 3. Sensor responses to the injection of 10− 10 M helional (A) and 10− 10 M heptanal (B). Measurements were performed in PBS pH 7.0 at 20 °C.
series with a parallel combination of the CPE (constant phase element with more or less capacitive characteristics depending on the biofilm behavior) and the Rp (interfacial polarization resistance in the absence of red-ox species). Table 1 presents the fitting values calculated in the ZView2 software (Scribner Associates, USA) for the sensor chips coated by gold whether bare or already modified with OR-based biofilm. As it can be seen, the ratio of CPE values of bare Au to CPE of modified Au is independent of temperature and remains about 4 ± 1. The n values of the modified electrodes are about 0.9 reflecting mainly the capacitive behavior of the corresponding CPE showing quite good insulating properties of the immobilized biofilms. 3.3. Impedance measurements in the presence of odorants at 20 °C Fig. 2. Impedance diagrams of OR-containing SAM-based biofilm under various polarization potentials of working electrode (vs SCE). Measurements were performed in PBS pH 7.0 at 20 °C.
In this experiment conducted at room temperature, aliquots of helional and heptanal were injected randomly into an electrochemical cell to the final concentrations 10− 11, 10− 10,
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Fig. 4. Polarization resistance Rp changes under stimulation of the OR 17–40 with helional or heptanal at 20 °C in PBS. Error bars are based on n = 2. Inset: Equivalent electrical circuit applied for the fitting of experimental impedance spectra.
10− 9, 10− 8 and 10− 7 M in PBS. The impedance spectrum was registered immediately after ligand injection. Then the surface was washed with PBS, stabilized during 10–15 min and used for the next odorant probing. Sensor response to 10− 10 M of helional at 20 °C is shown in Fig. 3A. Injection of the same aliquot of heptanal produced only a slight shift of the impedance spectrum as it can be seen from Fig. 3B. In order to control the signal specificity, a SAM-based biofilm without OR 17–40 was formed on the sensor chip and then exposed to helional and heptanal (from 10− 11 to 10− 7 M in PBS) at room temperature. All applied concentrations of both odorants produced insignificant variation of total impedance (data not shown). Obtained impedance data were fitted with the above-described electrical circuit in the frequency range 105–31 Hz. It was found that the injection of any ligand did affect significantly neither CPE nor n values, but the polarization resistance only, therefore the Rp shift was taken as a reporter of the OR 17–40 activity, i.e. as the sensor signal. Thus, the best signal was found for 10− 10 M helional, among the five concentrations tested in Fig. 4, where R0 is the fitting value of Rp before odorant injection, and R is the fitting value of Rp upon odorant stimulation of receptors. Stimulation of OR 17–40 with the blank solutions corresponding to 10− 11–10− 7 M helional resulted in a Rp shift ≤0.015. Therefore, the impedimetric sensor responses to 10− 9 and 10− 7 M helional and almost all responses to the heptanal should be considered as non-specific and/or insignificant. 3.4. Impedance measurements at 4 °C in the presence of odorants and GTP-γ-S It is known that the early steps of olfactory signal transduction in vertebrates involve the interaction of activated OR with G protein [22], which triggers the adenylate cyclase III catalyzed generation of cAMP thus leading to changes in membrane conductance [23–25]. Heterotrimeric G protein located on the cytoplasmic face of cell membrane possesses α, β and γ subunits [26]. Odorant-induced changes in OR
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conformation stimulate the replacement of GDP bound to Gα by GTP leading to Gα dissociation from βγ-dimer [26]. In such a way, desorption of Gα protein represents a biochemical signal by itself [11,18]. GTP-γ-S is a non-hydrolysable analogue of GTP recently reported to enhance an OR 17–40-based SPR sensor response to some odorants including helional [11]. So, the next step of our work was a more detailed analysis of OR 17–40 stimulation with various concentrations of odorants in the presence of GTP-γ-S in PBS at 4 °C. Two concentrations of helional, 10− 11 and 10− 7 M were probed at 4 °C in the absence of GTP-γ-S in order to know whether application of GTP-γ-S will enhance an impedimetric sensor response or not. Procedure of measurements in the presence of GTP-γ-S was as follows: at first, an electrochemical cell filled with PBS was cooled to 4 °C, then an aliquot of 1 mM GTP-γ-S PBS solution was injected to the final concentration 10 μM [11], and odorant was injected immediately after. All impedance spectra obtained at 4 °C were satisfactorily fitted with the above-described model circuit in the frequency range 4 × 103–2.5 Hz. While applied without GTP-γ-S, helional slightly altered the Rp parameter (Fig. 5A), but in the presence of GTP-γ-S the response to helional was higher (Fig. 5B). Fig. 6 demonstrates OR 17–40-based sensor responses to be 4 times higher in the presence of GTP-γ-S in the working buffer thus supporting a hypothesis about the enhancing role of GTP-γ-S in such a mode of odorant analysis. In addition, a oneorder increase of sensor sensitivity to helional was observed at 4 °C instead of 20 °C in the presence of GTP-γ-S. As it can be seen from Fig. 6, the maximal sensor response was already achieved after the injection of helional concentration 10− 11 M. In order to control whether an introduction of the GTP-γ-S also enhanced non-specific sensor responses, immobilized OR 17–40 was stimulated with blank solutions and heptanal (Fig. 7). Signals in response to the latter were found to be comparable with the alteration of Rp (∼0.025) caused by the blank probes in the presence of GTP-γ-S (Fig. 7). This non-specific shift Rp was 1.7 times larger at 4 °C instead of 20 °C, in the presence of GTP-γ-S. Therefore, the net advantage from the GTP-γ-S introduction should be estimated as a 2.3 fold increase in the specific sensor response. Since the stability of GTP-γ-S is known to be poor at 20 °C, odorant detection in the presence of GTP-γ-S at 20 °C was not attempted. Working at low temperature was found to increase the sensor lifetime from several hours (10–15 measurements at 20 °C) to Table 1 The fitting values calculated in ZView2 for the SPR chips coated with gold, bare and modified with OR 17–40 via SAM-based multilayer χ2 (× 10− 4)
Electrode
Frequency Temperature, Rs, CPE-T, μF n range, Hz °C Ω (rad/s)1−n
1. Bare Au 2. AuOR1740 3. Bare Au 4. Au-OR 1740
105–1.6
20
53 13.7
0.95
105–1.6
20
51
0.86 29328 11
4 × 103– 2.5 4 × 103– 2.5
4
57 19.9
0.91
4
86
0.92 19054 11
5.2
3.4
Rp, Ω 9350
1319
9
8
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Fig. 7. Polarization resistance Rp changes under stimulation of OR 17–40 with heptanal and blank probes at 4 °C in PBS with 10 μM GTP-γ-S. Error bars are based on n = 2.
2 days of continuous work at 4 °C. The most evident reason of this phenomenon is better preservation of receptor and/or G protein proper conformation which is of high importance for the intrinsic activity of these proteins. 3.5. Modeling the impact of nanosomes size on a sensitive layer formation
Fig. 5. Sensor responses to 10− 11 M helional. Measurements were conducted at 4 °C in the absence (A) or presence (B) of 10 μM GTP-γ-S in PBS pH 7.0.
Fig. 6. Polarization resistance Rp changes under stimulation of the OR 17–40 with helional at 4 °C in PBS with 10 μM GTP-γ-S. Error bars are based on n = 2. Inset: Polarization resistance Rp changes under stimulation of the OR 1740 with helional in the presence or absence of GTP-γ-S in PBS at 4 °C.
In Section 3.1 we remarked a slight but evident difference of 15% in the average SPR signals to adsorption of the membrane vesicles sonicated during 10 and 20 min. Since the duration of suspension sonication apparently correlates to its homogeneity, one can suggest that the dispersity of vesicles size could affect their immobilization. In order to calculate the possible impact of nanosomes size on their surface coverage factor (SCF) we have used the Monte-Carlo simulation. Here the SCF is the ratio of an electrode surface area
Fig. 8. Monte-Carlo simulation of nanosomes distribution on the flat electrode surface. The present result was obtained using the following parameters values: electrode diameter: 100 a.u., nanosome diameter: 1 a.u., dispersity of nanosomes diameters: 0% or 80%, number of placement attempts: 1000, number of modeling cycles: 10,000.
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covered with nanosomes to a total surface accessible for specific anchoring of membrane vesicles and it evidently has to correlate positively with the SPR signal variation. The following assumptions were used for the simulation: i) all nanosomes were of the same shape (spheres) which could vary in size (diameter). Two cases were considered: all nanosomes had the same diameter (dispersity of sizes is equal to 0%), or various diameters (dispersity of sizes is equal to 80% like in the suspension of vesicles from 50 to 500 nm); ii) electrode surface was considered as a flat disk, whose diameter was much larger than that of a single nanosome; iii) fixed number of attempts was used to place a nanosome on the electrode surface. If no attempt was successful, the electrode was considered as optimally covered by nanosomes. The result (Fig. 8) of this simulation demonstrates that the wider the dispersity of the nanosomes sizes, the larger the area covered. It is obvious that the absolute value of vesicles diameter does not impact the SCF (Fig. 8), therefore, only varying the dispersity of the nanosomes diameters can one increase the SCF and the amount of olfactory receptor on the surface as well. So, the above-described model can explain a more efficient adsorption of the nanosomes obtained after only 10 min of sonication, however, it is not evident whether it will enhance or restrict the biofilm sensitivity to odorant, and this has to be studied. 4. Conclusion Layer-by-layer formation of the OR 17–40-based biofilm on the SPR sensor chip was proven to be an efficient and wellreproducible process. Under injections of helional resulting in submicromolar to subnanomolar concentrations, activation of OR 17–40 was electrochemically revealed. Applying GTP-γ-S at 4 °C as a specific enhancer of biochemical signal, it became possible to detect 10− 11 M helional. The low temperature improved the lifetime of olfactory biosensor up to 2 days of continuous work. Next steps of our work with human olfactory receptor OR 17–40 will include testing of broader range of odorants of various concentrations. Already obtained results provide a promising basis for the usability of olfactory receptors in the construction of labelfree biosensors as elements of a bioelectronic nose. Acknowledgements This work was financially supported by the SPOT-NOSED project (IST-2001-38739).
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Materials Science and Engineering C 28 (2008) 633 – 639 www.elsevier.com/locate/msec
Electrochemical study of human olfactory receptor OR 17–40 stimulation by odorants in solution I.V. Benilova a,c,⁎, J. Minic Vidic b , E. Pajot-Augy b , A.P. Soldatkin c , C. Martelet a , N. Jaffrezic-Renault d,1 b
a Laboratoire AMPERE, UMR-CNRS 5005, Ecole Centrale de Lyon, 69134 Ecully Cedex, France INRA, Neurobiologie de l'Olfaction et de la Prise Alimentaire, Récepteurs et Communication Chimique, Domaine de Vilvert, 78352 Jouy-en-Josas Cedex, France c Laboratory of Biomolecular Electronics, Institute of Molecular Biology and Genetics NASU, 03143 Kyiv, Ukraine d Université Claude Bernard—Lyon 1, Laboratoire des Sciences Analytiques, UMR-CNRS 5180, 69622 Villeurbanne Cedex, France
Available online 13 October 2007
Abstract The human olfactory receptor OR 17–40 co-expressed with α-subunit of Golf protein in yeast was attempted as a bio recognition part of impedimetric biosensor. The receptor in its natural membrane environment was anchored to a gold-coated glass substrate modified with thiolbased multilayer. Step-by-step building-up of the OR 17–40 based biofilm was monitored using surface plasmon resonance technique. Stimulation of the OR 17–40 with its cognate odorant helional in phosphate-buffered saline was probed by means of electrochemical impedance spectroscopy under various conditions. Activation of OR 17–40 in the presence of GTP-γ-S at 4 °C was found to improve the sensitivity of the developed labelfree biosensor, probably via the enhancement of the specific biochemical signal. © 2008 Elsevier B.V. All rights reserved. Keywords: Impedance spectroscopy; SPR; Bioelectronic nose; Human olfactory receptor; Helional
1. Introduction Simultaneous detection of different odorants in liquid and gaseous mixtures is extremely important for quality control in food, beverage and fragrance industries. Nowadays, the instruments capable of a broad-band routine analysis of complex aroma are mainly represented by mass spectrometers, or gas chromatographs combined to mass spectrometers (GC/MS). However, a spectrum of odor peaks obtained by means of GC/MS does not actually provide sufficient information about aroma quality itself which, however, directly arises from the human odor impressions [1]. The arrays of semiselective sensors coupled to the patternrecognition systems and called artificial (electronic) noses [1–3] ⁎ Corresponding author. Université Claude Bernard—Lyon 1, bât. Jules Raulin, 5ème étage, Laboratoire des Sciences Analytiques, UMR-CNRS 5180, 69622 Villeurbanne Cedex, France. Tel.: +33 4 72 43 11 82; fax: +33 4 72 43 12 06. E-mail address: [email protected] (I.V. Benilova). 1 Université Claude Bernard—Lyon 1, bât. Jules Raulin, 5ème étage, Laboratoire des Sciences Analytiques, UMR-CNRS 5180, 69622 Villeurbanne Cedex, France. 0928-4931/$ - see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2007.10.040
present another kind of analytical instrumentation that mimics natural olfactory system operation. As sensitive elements of electronic noses, conductive organic polymers [4,5], porphyrins [6], calixarenes [7–9], can be used. In the last years, the curiosity of scientists to achieve a deeper understanding of the functional peculiarities of olfactory system of vertebrates has stimulated the development of a bioelectronic nose [1,10]—multisensor device whose biorecognition part consists in natural G protein coupled olfactory receptors. Many recent reports concern the elaboration of individual sensors based on the ORs expressed either in olfactory sensory neurons [10] or in heterologous cells [11–14] and coupled to various transducers like quartz-crystal microbalance [13,14], lightaddressable potentiometric sensor [10], substrates for the SPR (surface plasmon resonance) [11] and the EIS (electrochemical impedance spectrometry) [12] techniques. Some authors consider the human olfactory receptors to be narrowly selective for the individual odorants in contrast to the rodent ORs which are able to respond to a large repertoire of ligands [15]. However, the sensitivity of human nose does not
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arise from the values of binding constants of odorant-receptor coupling itself but rather from the signal processing in the higher brain structures [2], and therefore a choice of appropriate data processing model is an extremely important step in the bioelectronic nose elaboration. Artificial neural networks are one of the most widely used pattern-recognition models [16,17] dealing with complex nonlinear dose-dependence relations and therefore can be very suitable for the bioelectronic noses. However, dealing with time-consuming complex training of such networks requires very high stability of ORs coupled to the transducers and a satisfactory low level of stochastic responses as well. Previously, our group has studied the sensitivity of heterologously expressed rat OR I7 [12] and human OR 17–40 [11] towards a set of different odorants, by means of the EIS and the SPR technique, respectively. In this work, for the first time, a stimulation of SPR chip-coupled OR 17–40 by two ligands, preferential (helional) and unrelated (heptanal), was probed by means of nonfaradaic EIS. In order to improve a resolution of such an impedimetric label-free sensor via the enhancement of the biochemical signal, ligand-receptor interactions were also investigated in the presence of the guanosine triphosphate (GTP-γ-S). 2. Materials and methods 2.1. Biomaterials and chemicals Gαolf protein and human OR 17–40 tagged with cmyc sequence on its N-terminus were co-expressed in yeast Saccharomyces cerevisiae (strain MC18) [18] and the membrane fraction was prepared as described in [11]. Stock suspension of membrane fraction with protein content 2.85 mg/ml was aliquoted and frozen at − 80 °C. Monoclonal anti-cmyc antibody (Ab) was obtained from Roche Molecular Biochemical and biotinylated by means of DSB-X™ Biotin Protein Labeling Kit (Molecular Probes, Leiden, Netherlands). Stock solution of Ab (2.55 mg/ml) was divided into aliquots and stored at −20 °C. 16-Mercaptohexadecanoic acid (90% purity) and 1,2dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-biotinyl sodium salt (biotinyl-PEA) were purchased, respectively, from Aldrich and Avanti Polar Lipids. GTP-γ-S (guanosine-5′-O-(3thiotriphosphate); MW 563, 93% purity), bovine serum albumin (BSA; 98% purity) and goat IgG were obtained from Sigma, neutravidin—from Pierce. Ethanol (99.8%), HCl (37%), HNO3 (65%), H2O2 (30%) and NH4OH (25%) were purchased from Fluka and used as received. Dimethyl sulfoxide (DMSO) for odorant dilution and heptanal were obtained from Sigma. Helional was a kind gift from Givaudan-Roure (Switzerland). As a working buffer, a phosphate-buffered saline (PBS) was used with the following composition: 8 mM Na2HPO4, 1.5 mM KH2PO4, 3 mM KCl, 150 mM NaCl, pH 7.0 [19]. All reagents for PBS preparation were of analytical grade. All aqueous solutions were prepared with ultrapure water from Milli-Q system.
2.2. SPR technique and gold-coated substrates Surface plasmon resonance spectrometer “Biosuplar 3” (www.micro-systems.de) was developed at the Institute of Semiconductor Physics of the National Academy of Sciences of Ukraine (Kyiv). This optoelectronic device based on the phenomenon of the surface plasmon resonance in the Kretchmann's optical configuration was controlled by a computer via self-developed software. Gold film (45 nm) deposited through Cr adhesion layer (1–1.5 nm) onto a glass chip represented the sensor surface. An incident beam of ppolarized light from a semiconductor laser diode (λ = 650 nm) excited an oscillation of electronic plasma (i.e. surface plasmon) in this metallic film. A special prism capable of rotation on a computer-defined angle provided optimal conditions for the plasmon excitation. A plasmon resonance itself was registered as a drastic decrease of reflected light intensity. In the present study, the SPR spectrometer flow cell (V∼20 μl) was connected to a Gilson Minipuls 3 pump. 2.3. Pretreatment of sensor surface Before work, substrates were cleaned by a mixture “aqua regia” (H2O + HCl + HNO3, 16 + 3 + 1, v/v) during 1–1.5 min and then—with a basic mixture (H2O + H2O2 + NH4OH, 5 + 1 + 1, v/v) during 15 s. Thoroughly rinsed with water, every chip was then immersed into ethanol for several seconds. 2.4. Self-assembly of the mixed layer onto gold To obtain mixed self-assembled monolayer (SAM) on gold surface, 1 mM mercaptohexadecanoic acid and 0.1 mM biotinyl-PEA dissolved in ethanol were incubated with freshly cleaned chip during 21 h [20]. Mercaptohexadecanoic acid is fixed onto Au via the chemisorption of SH-groups, as to biotinyl-PEA, it can be inserted between long-chain thiols due to the numerous hydrophobic interactions. Such mixed SAM provides a good basis for the further anchoring of biomolecules to the chip surface. To elute unfixed molecules, chip was rinsed with ethanol, and dried under a nitrogen flow. 2.5. Blocking step and formation of the upper supporting layers In order to saturate all non-specific adsorption sites on SAM surface, heterogeneous layer was treated by 1 mM solution either of IgG or BSA in PBS in the flow cell: 0.3 ml of blocking molecules solution was run on the chip at the flow rate (FR) 0.04 ml/min. Neutravidin (0.5 μM, 0.3 ml solution in PBS) and biotinylated anti-cmyc Ab (0.5 μM, 0.3 ml solution in PBS) were subsequently run on the blocked SAM at the FR 0.04 ml/min. Before the formation of any upper molecular layer, the previous one was rinsed with PBS during 5–15 min. 2.6. Preparation and immobilization of OR 17–40 Stock suspension of OR 17–40 in its natural membrane fraction was thawed and resuspended in PBS on ice up to the
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OR concentration 70 μg/ml. 0.3 ml of this suspension was treated in the ultrasonic bath Elmasonic (35 kHz, 120 W) in icecold water during 10 or 20 min in order to obtain more or less heterogeneous suspension of membrane nanovesicles [11]. Afterwards, 0.3 ml of this suspension was immediately run on the chip (FR 0.04 ml/min). 2.7. Electrochemical probing After immobilization of the OR 17–40, SPR chip was washed with PBS and transferred into a three-electrode electrochemical glass cell (V = 5 ml) placed into a Faradaic cage and filled with PBS. The EIS measurements were performed with an impedance spectrometer Voltalab 40. In order to conduct measurements at 4 ± 0.5 °C, the cell was connected to the circulator thermostat Julabo F25 (France). All other measurements were performed at room temperature (20 ± 1 °C). As a reference a saturated calomel electrode (SCE) from Radiometer Analytical was employed, and a platinum plate was used as an auxiliary electrode. Active surface area of the latter was 0.64 cm2, and 0.25 cm2 for the working electrode (i.e. biofunctionalized chip). Impedance was swept in the frequency range 105–0.1 Hz at dc potential − 700 mV vs SCE with superimposed excitation voltage of 10 mV. Data were visualized as Cole–Cole (Nyquist) plots via the VoltaMaster 4.0 software. 2.8. Preparation of odorant solutions Only freshly prepared solutions of helional and heptanal were used in all experiments. Stock 0.1 M solutions were prepared by the dilution of corresponding aldehyde in DMSO, but further dilutions were performed only in PBS. 10− 3 M dilution was directly prepared from 0.1 M, then further dilutions were obtained by successive 1:10 dilutions. To take into account a possible solvent effect in biosensor response, blank probes at the various dilutions were prepared, replacing the helional by PBS. 2.9. Monte-Carlo simulation In order to estimate the impact of nanosomes size on their efficient immobilization, a Monte-Carlo simulation was used. A correspondent model was created using the MATLAB 7.0 software. 3. Results and discussion 3.1. Layer-by-layer assembly of OR-containing biofilm A typical kinetics of step-by-step formation of the ORcontaining biofilm is shown in Fig. 1A. At first, PBS was run on the chip surface functionalized with SAM; afterwards it was treated with IgG or BSA. Adsorption of BSA onto the surface produced a signal shift of about 0.12 arc degrees (Fig. 1A), while the response to the IgG anchoring was about 2.5 times higher (data not shown), probably due to the difference in the molecular mass of BSA (66 kDa) and IgG (150 kDa).
Fig. 1. (A) Typical building-up kinetics of the OR-containing SAM-based biofilm. (B) SPR responses to the step-by-step formation of OR-containing multilayer. All measurements were performed in the flow cell in PBS at pH 7.0.
The next step of surface modification consisted in the immobilization of neutravidin. Neutravidin (60 kDa) presents an interesting alternative to the streptavidin since it is a neutral protein without any carbohydrate moieties, thus possessing rather low capabilities of non-specific binding at physiological pH [21]. Neutravidin molecule has 4 binding sites (two on each opposite sides), presenting a very suitable linker for the consecutive anchoring of any biotinylated molecules. As it can be seen from Fig. 1B, anchoring of neutravidin to the BSAblocked or IgG-blocked SAM produces a resonance angle shift of about 0.2–0.25 arc degrees. However, coupling of neutravidin to the non-blocked SAM produced a higher signal presumably due to some spatial restriction of accessibility of biotin groups after BSA/IgG blockage step. Biotinylated anti-cmyc Ab was immobilized over the neutravidin layer. Anchoring of Ab onto the surface blocked either with IgG or BSA produced a signal of about 0.3 arc degrees being 1.7 times weaker than it could be expected from the molecular mass ratio Ab : neutravidin = 2.5. Maximal SPR response to Ab anchoring (0.55 arc degrees) was achieved on the non-blocked neutravidin layer, however, the average binding
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capabilities of the latter were shown to be statistically the same as those of the BSA and IgG-blocked structures (Fig. 1B). Finally, a suspension of OR 17–40-containing nanosomes was run on the surface and incubated for about 20–30 min, then rinsed with PBS. The best average SPR signal for the OR 17–40 immobilization was obtained on the basis of the BSA-blocked structure (Fig. 1B). The latter was even more efficient than the non-blocked structure demonstrating a good accessibility and a proper orientation of the anti-cmyc binding sites of Ab. As to the IgG-blocked multilayer, it was 3 times less capable of nanosomes binding than the biofilm blocked with BSA. The impact of ultrasonication duration on the nanosomes adsorption on the BSA-blocked surfaces was also investigated. It was found that nanovesicles pretreated during 10 min produced the SPR signal of 0.26 ± 0.07 arc degrees (n = 8), while those sonicated during 20 min yielded 0.22 ± 0.03 arc degrees (n = 5). So, the SPR chips modified by the BSA-blocked multilayer coated with OR-containing nanosomes sonicated during 10 min were chosen for further electrochemical studies. A step-by-step formation of OR-containing biofilm took about 280–330 min and was a fully real time controllable process. 3.2. Impedance probing of SPR sensor chips After the OR-containing biofilm formation, the SPR chip was employed as the working electrode in the electrochemical cell. Multilayer revealed a high stability upon contact with PBS solution and rather high complex impedance as well (Fig. 2). As a working potential, − 700 mV vs SCE was chosen and applied for all measurements. Under this potential the OR-containing biofilm remained stable in time, and crude complex impedance data (e.g., like in Figs. 3 (A,B) and 5 (A,B)) gave a good fit to an electrical circuit shown in Fig. 4 over the broad frequency range. The above-mentioned circuit taken for data processing is composed of the ohmic resistance of bulk electrolyte (Rs) in
Fig. 3. Sensor responses to the injection of 10− 10 M helional (A) and 10− 10 M heptanal (B). Measurements were performed in PBS pH 7.0 at 20 °C.
series with a parallel combination of the CPE (constant phase element with more or less capacitive characteristics depending on the biofilm behavior) and the Rp (interfacial polarization resistance in the absence of red-ox species). Table 1 presents the fitting values calculated in the ZView2 software (Scribner Associates, USA) for the sensor chips coated by gold whether bare or already modified with OR-based biofilm. As it can be seen, the ratio of CPE values of bare Au to CPE of modified Au is independent of temperature and remains about 4 ± 1. The n values of the modified electrodes are about 0.9 reflecting mainly the capacitive behavior of the corresponding CPE showing quite good insulating properties of the immobilized biofilms. 3.3. Impedance measurements in the presence of odorants at 20 °C Fig. 2. Impedance diagrams of OR-containing SAM-based biofilm under various polarization potentials of working electrode (vs SCE). Measurements were performed in PBS pH 7.0 at 20 °C.
In this experiment conducted at room temperature, aliquots of helional and heptanal were injected randomly into an electrochemical cell to the final concentrations 10− 11, 10− 10,
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Fig. 4. Polarization resistance Rp changes under stimulation of the OR 17–40 with helional or heptanal at 20 °C in PBS. Error bars are based on n = 2. Inset: Equivalent electrical circuit applied for the fitting of experimental impedance spectra.
10− 9, 10− 8 and 10− 7 M in PBS. The impedance spectrum was registered immediately after ligand injection. Then the surface was washed with PBS, stabilized during 10–15 min and used for the next odorant probing. Sensor response to 10− 10 M of helional at 20 °C is shown in Fig. 3A. Injection of the same aliquot of heptanal produced only a slight shift of the impedance spectrum as it can be seen from Fig. 3B. In order to control the signal specificity, a SAM-based biofilm without OR 17–40 was formed on the sensor chip and then exposed to helional and heptanal (from 10− 11 to 10− 7 M in PBS) at room temperature. All applied concentrations of both odorants produced insignificant variation of total impedance (data not shown). Obtained impedance data were fitted with the above-described electrical circuit in the frequency range 105–31 Hz. It was found that the injection of any ligand did affect significantly neither CPE nor n values, but the polarization resistance only, therefore the Rp shift was taken as a reporter of the OR 17–40 activity, i.e. as the sensor signal. Thus, the best signal was found for 10− 10 M helional, among the five concentrations tested in Fig. 4, where R0 is the fitting value of Rp before odorant injection, and R is the fitting value of Rp upon odorant stimulation of receptors. Stimulation of OR 17–40 with the blank solutions corresponding to 10− 11–10− 7 M helional resulted in a Rp shift ≤0.015. Therefore, the impedimetric sensor responses to 10− 9 and 10− 7 M helional and almost all responses to the heptanal should be considered as non-specific and/or insignificant. 3.4. Impedance measurements at 4 °C in the presence of odorants and GTP-γ-S It is known that the early steps of olfactory signal transduction in vertebrates involve the interaction of activated OR with G protein [22], which triggers the adenylate cyclase III catalyzed generation of cAMP thus leading to changes in membrane conductance [23–25]. Heterotrimeric G protein located on the cytoplasmic face of cell membrane possesses α, β and γ subunits [26]. Odorant-induced changes in OR
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conformation stimulate the replacement of GDP bound to Gα by GTP leading to Gα dissociation from βγ-dimer [26]. In such a way, desorption of Gα protein represents a biochemical signal by itself [11,18]. GTP-γ-S is a non-hydrolysable analogue of GTP recently reported to enhance an OR 17–40-based SPR sensor response to some odorants including helional [11]. So, the next step of our work was a more detailed analysis of OR 17–40 stimulation with various concentrations of odorants in the presence of GTP-γ-S in PBS at 4 °C. Two concentrations of helional, 10− 11 and 10− 7 M were probed at 4 °C in the absence of GTP-γ-S in order to know whether application of GTP-γ-S will enhance an impedimetric sensor response or not. Procedure of measurements in the presence of GTP-γ-S was as follows: at first, an electrochemical cell filled with PBS was cooled to 4 °C, then an aliquot of 1 mM GTP-γ-S PBS solution was injected to the final concentration 10 μM [11], and odorant was injected immediately after. All impedance spectra obtained at 4 °C were satisfactorily fitted with the above-described model circuit in the frequency range 4 × 103–2.5 Hz. While applied without GTP-γ-S, helional slightly altered the Rp parameter (Fig. 5A), but in the presence of GTP-γ-S the response to helional was higher (Fig. 5B). Fig. 6 demonstrates OR 17–40-based sensor responses to be 4 times higher in the presence of GTP-γ-S in the working buffer thus supporting a hypothesis about the enhancing role of GTP-γ-S in such a mode of odorant analysis. In addition, a oneorder increase of sensor sensitivity to helional was observed at 4 °C instead of 20 °C in the presence of GTP-γ-S. As it can be seen from Fig. 6, the maximal sensor response was already achieved after the injection of helional concentration 10− 11 M. In order to control whether an introduction of the GTP-γ-S also enhanced non-specific sensor responses, immobilized OR 17–40 was stimulated with blank solutions and heptanal (Fig. 7). Signals in response to the latter were found to be comparable with the alteration of Rp (∼0.025) caused by the blank probes in the presence of GTP-γ-S (Fig. 7). This non-specific shift Rp was 1.7 times larger at 4 °C instead of 20 °C, in the presence of GTP-γ-S. Therefore, the net advantage from the GTP-γ-S introduction should be estimated as a 2.3 fold increase in the specific sensor response. Since the stability of GTP-γ-S is known to be poor at 20 °C, odorant detection in the presence of GTP-γ-S at 20 °C was not attempted. Working at low temperature was found to increase the sensor lifetime from several hours (10–15 measurements at 20 °C) to Table 1 The fitting values calculated in ZView2 for the SPR chips coated with gold, bare and modified with OR 17–40 via SAM-based multilayer χ2 (× 10− 4)
Electrode
Frequency Temperature, Rs, CPE-T, μF n range, Hz °C Ω (rad/s)1−n
1. Bare Au 2. AuOR1740 3. Bare Au 4. Au-OR 1740
105–1.6
20
53 13.7
0.95
105–1.6
20
51
0.86 29328 11
4 × 103– 2.5 4 × 103– 2.5
4
57 19.9
0.91
4
86
0.92 19054 11
5.2
3.4
Rp, Ω 9350
1319
9
8
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Fig. 7. Polarization resistance Rp changes under stimulation of OR 17–40 with heptanal and blank probes at 4 °C in PBS with 10 μM GTP-γ-S. Error bars are based on n = 2.
2 days of continuous work at 4 °C. The most evident reason of this phenomenon is better preservation of receptor and/or G protein proper conformation which is of high importance for the intrinsic activity of these proteins. 3.5. Modeling the impact of nanosomes size on a sensitive layer formation
Fig. 5. Sensor responses to 10− 11 M helional. Measurements were conducted at 4 °C in the absence (A) or presence (B) of 10 μM GTP-γ-S in PBS pH 7.0.
Fig. 6. Polarization resistance Rp changes under stimulation of the OR 17–40 with helional at 4 °C in PBS with 10 μM GTP-γ-S. Error bars are based on n = 2. Inset: Polarization resistance Rp changes under stimulation of the OR 1740 with helional in the presence or absence of GTP-γ-S in PBS at 4 °C.
In Section 3.1 we remarked a slight but evident difference of 15% in the average SPR signals to adsorption of the membrane vesicles sonicated during 10 and 20 min. Since the duration of suspension sonication apparently correlates to its homogeneity, one can suggest that the dispersity of vesicles size could affect their immobilization. In order to calculate the possible impact of nanosomes size on their surface coverage factor (SCF) we have used the Monte-Carlo simulation. Here the SCF is the ratio of an electrode surface area
Fig. 8. Monte-Carlo simulation of nanosomes distribution on the flat electrode surface. The present result was obtained using the following parameters values: electrode diameter: 100 a.u., nanosome diameter: 1 a.u., dispersity of nanosomes diameters: 0% or 80%, number of placement attempts: 1000, number of modeling cycles: 10,000.
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covered with nanosomes to a total surface accessible for specific anchoring of membrane vesicles and it evidently has to correlate positively with the SPR signal variation. The following assumptions were used for the simulation: i) all nanosomes were of the same shape (spheres) which could vary in size (diameter). Two cases were considered: all nanosomes had the same diameter (dispersity of sizes is equal to 0%), or various diameters (dispersity of sizes is equal to 80% like in the suspension of vesicles from 50 to 500 nm); ii) electrode surface was considered as a flat disk, whose diameter was much larger than that of a single nanosome; iii) fixed number of attempts was used to place a nanosome on the electrode surface. If no attempt was successful, the electrode was considered as optimally covered by nanosomes. The result (Fig. 8) of this simulation demonstrates that the wider the dispersity of the nanosomes sizes, the larger the area covered. It is obvious that the absolute value of vesicles diameter does not impact the SCF (Fig. 8), therefore, only varying the dispersity of the nanosomes diameters can one increase the SCF and the amount of olfactory receptor on the surface as well. So, the above-described model can explain a more efficient adsorption of the nanosomes obtained after only 10 min of sonication, however, it is not evident whether it will enhance or restrict the biofilm sensitivity to odorant, and this has to be studied. 4. Conclusion Layer-by-layer formation of the OR 17–40-based biofilm on the SPR sensor chip was proven to be an efficient and wellreproducible process. Under injections of helional resulting in submicromolar to subnanomolar concentrations, activation of OR 17–40 was electrochemically revealed. Applying GTP-γ-S at 4 °C as a specific enhancer of biochemical signal, it became possible to detect 10− 11 M helional. The low temperature improved the lifetime of olfactory biosensor up to 2 days of continuous work. Next steps of our work with human olfactory receptor OR 17–40 will include testing of broader range of odorants of various concentrations. Already obtained results provide a promising basis for the usability of olfactory receptors in the construction of labelfree biosensors as elements of a bioelectronic nose. Acknowledgements This work was financially supported by the SPOT-NOSED project (IST-2001-38739).
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