Biosensor Device and Methods

Abstract

The invention provides a sensor device comprising an insect odorant receptor complex, comprising an OrX and an Oreo, in electrical communication with a substrate, wherein the sensor device is configured to detect a change in an electrical characteristic of the substrate. The invention also provides sensor device component comprising an insect odorant receptor complex, comprising an OrX and an Oreo, in electrical communication with a substrate. The invention also provides methods for manufacture and use of the sensor device and sensor device component. The invention also provides methods of use of the sensor to detect an analyte.

Claims

1. A sensor device comprising an insect odorant receptor complex, comprising an OrX and an Orco, in electrical communication with a substrate, wherein the sensor device is configured to detect a change in an electrical characteristic of the substrate.

2. The sensor device of claim 1 in which the change in the electrical characteristic results from an interaction between the OrX in the insect odorant receptor complex, and an analyte.

3. The sensor of any preceding claim in which the sensor is capable of detecting binding of an analyte to the OrX in the insect odorant receptor complex by detecting the change in the electrical characteristic of the substrate.

4. The sensor of any preceding claim in which the insect odorant receptor complex is present in a form that is capable of undergoing a conformational change in response binding of the analyte.

5. The sensor of any one of claims 1 to 3 wherein the insect odorant receptor complex forms an ion channel sensitive to the presence or otherwise of the analyte.

6. The sensor of any preceding claim in which the insect odorant receptor complex is present in a membrane mimic.

7. The sensor of claim 6 in which membrane mimic is selected from a liposome, an amphipole, a detergent micelle, a nanovesicle, a lipid bilayer, a nanodisc, and a surfactant.

8. The sensor of claim 5 or claim 6, in which the membrane mimic comprises amphipathic molecules such as lipid molecules, preferably wherein the amphipathic molecules comprise phospholipid molecules.

9. The sensor of claim 3 in which the sensor can detect the presence of the analyte at a concentration of less than 1×10.sup.−3M, preferably wherein the sensor can detect the presence of the analyte at a concentration of less than 1×10.sup.−9M.

10. The sensor of claim 9 in which the sensor the sensor can detect the presence of the analyte at a concentration of less than 1×10.sup.−12 M, preferably wherein the sensor can detect the presence of the analyte at a concentration of less than 1×10.sup.−15 M.

11. The sensor of any preceding claim in which the substrate is selected from, or composed of, at least one of: an electrode, a semiconductor material, carbon nanotubes (CNTs), graphene, an oxide, doped silicon, a conducting polymer, and a resonator component.

12. The sensor of any preceding claim in which the electrical characteristic is selected from at least one of: conductivity, resistance, complex resistance, impedance, electrochemical impedance, electrochemical potential, the flow of current, and the resonance frequency of oscillations induced by an alternating electric field.

13. The sensor of any preceding claim in which the sensor comprises: a membrane mimic which comprises amphipathic molecules, an OrX protein and an Orco protein; a first substrate which comprises a first electrode disposed at a first side of the membrane; and a second substrate which comprises a second electrode disposed at a second side of the membrane.

14. The sensor of claim 14, wherein the sensor comprises a control system which is configured to measure an electrical characteristic across the first and second electrodes, preferably to detect a current flowing between the first and second electrodes.

15. A method of detecting an analyte, the method comprising the steps: a) binding of the analyte to the insect OrX in the sensor of any preceding claim, b) detecting a change in an electrical characteristic of the substrate, wherein the change in the electrical characteristic of the substrate indicates detection of the analyte.

16. A method of detecting the presence of an analyte in an environment, the method comprising the steps: a) exposing the sensor of any preceding claim to an environment containing the analyte, b) binding of the analyte to the insect OrX in the sensor c) detecting a change in an electrical characteristic of the substrate, wherein the change in the electrical characteristic of the substrate indicates presence of the analyte in the environment.

17. A method of manufacturing a sensor device the method including the step of establishing electrical communication between an insect odorant receptor complex, comprising an OrX and an Orco, and the substrate of the sensor device, wherein the sensor device is configured to detect a change in an electrical characteristic of the substrate.

18. A sensor device component comprising an insect odorant receptor complex, comprising an OrX and an Orco, in electrical communication with a substrate.

19. A sensor device comprising the component of claim 13, wherein the sensor device is configured to detect a change in an electrical characteristic of the substrate.

20. A method of manufacturing a sensor device component, the method including the step of establishing electrical communication between an insect odorant receptor complex, comprising an OrX and an Orco, and a substrate.

21. A method of assembling a sensor device, the method comprising adding sensor device component of claim 13 to the sensor device, wherein the assembled sensor device is configured to detect a change in an electrical characteristic of the substrate.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0676] The present invention will be better understood with reference to the accompanying non-limiting drawings in which:

[0677] FIG. 1. Schematic representation of the insect OR membrane complex, comprised of an odorant binding OrX subunit and an Orco subunit to produce a ligand-gated non-selective cation channel. The orange circle represents the bound odorant.

[0678] FIG. 2. Schematic representation of the EIS sensor preparation starting with electrode cleaning, followed by SAM formation and completed with the covalent attachment of liposomes onto the SAM layer. Electrochemical read-out is obtained from EIS measurements carried out in three terminal electrochemical set-ups. Circles with blue shape represent liposome-integrated insect OrXs or OrX/Orco complexes, and red shapes represent VOC ligands.

[0679] FIG. 3 shows AFM height images (a-d), roughness profile indicated by the marked line on height images (e-h), and 3D images (i-l) of bare, SAM modified, NHS-EDC coupled, and Or22a/liposomes immobilized gold surfaces, respectively.

[0680] FIG. 4. (A) Impedance evolution of an Or10a liposomes functionalised electrode versus the target ligand methyl salicylate (metsal) with a concentration range from 1 aM to 100 μM. Experimental data are presented as symbols and the equivalent circuit fitting curves as solid lines. (B) Dose response curve for gold electrodes functionalized with Or10a liposomes in response to the target ligand methyl salicylate, and control ligand methyl hexanoate. The target and control ligand binding measurements were also performed with gold electrodes functionalised with empty liposomes. Error bars were generated using standard deviation using four repeats.

[0681] FIG. 5. (A) Impedance evolution of an Or10a/Orco liposomes functionalised electrode versus the target ligand methyl salicylate (metsal) with a concentration range from 1 aM to 100 μM. Experimental data are presented as symbols and the equivalent circuit fitting curves as solid lines. (B) Dose response curve for gold electrodes functionalized with Or10a/Orco liposomes in response to the target ligand Methyl salicylate. The target ligand binding measurements were also performed with gold electrodes functionalised with empty liposomes and Orco liposomes demonstrating a null response. Error bars were generated using standard deviation using four repeats.

[0682] FIG. 6. (A) Impedance evolution of an Or22a liposomes functionalised electrode versus the target ligand methyl hexanoate (methex) with a concentration range from 1 aM to 100 μM. Experimental data are presented as symbols and the equivalent circuit fitting curves as solid lines. (B) Dose response curve for gold electrodes functionalized with Or22a liposomes in response to the target ligand methyl hexanoate, and control ligand methyl salicylate. The target and control ligand binding measurements were also performed with gold electrodes functionalised with empty liposomes. Error bars were generated using standard deviation using four repeats.

[0683] FIG. 7. (A) Impedance evolution of an Or22a/Orco liposomes functionalised electrode versus the target ligand methyl hexanoate (methex) with a concentration range from 1 aM to 100 μM. Experimental data are presented as symbols and the equivalent circuit fitting curves as solid lines. (B) Dose response curve for gold electrodes functionalized with Or22a/Orco liposomes in response to the target ligand methyl hexanoate. The target ligand binding measurements were also performed with gold electrodes functionalised with empty liposomes and Orco liposomes demonstrating a null response. Error bars were generated using standard deviation using four repeats.

[0684] FIG. 8. (A) Impedance evolution of (A) an Or35a in liposomes functionalised electrode versus the target ligand E2-hexenal with a concentration range from 1 aM to 100 μM. Experimental data are presented as symbols and the equivalent circuit fitting curves as solid lines. (B) Dose response curve for gold electrodes functionalized with Or35a liposomes in response to the target ligand E2-hexenal, and control ligand methyl salicylate. The target and control ligand binding measurements were also performed with gold electrodes functionalised with empty liposomes. Error bars were generated using standard deviation using four repeats.

[0685] FIG. 9. (A) Impedance evolution of (A) an Or35a/Orco in liposomes functionalised electrode versus the target ligand E2-hexenal with a concentration range from 1 aM to 100 μM. Experimental data are presented as symbols and the equivalent circuit fitting curves as solid lines. B) Dose response curve for gold electrodes functionalized with Or35a/Orco liposomes in response to the target ligand E2-hexenal. The target ligand binding measurements were also performed with gold electrodes functionalised with empty liposomes and Orco liposomes demonstrating a null response. Error bars were generated using standard deviation using four repeats.

[0686] FIG. 10. Summary of EIS dose response curves for (a) Or10a+/−Orco, (b) Or22a+/−Orco, and (c) Or35a+/−Orco liposomes in response to target and control ligands. All three dose response curves show that the presence of Orco causes each OrX receptor to bind its target compound with greater sensitivity, thus shifting the dose response curve to the left. Error bars were generated using standard deviation using four repeats.

[0687] FIG. 11. (A) The change in frequency on the Quartz crystal microbalance with Dissipation (QCM-D) with SAM and NHS/EDC modification, Or10a liposome (top) or Or10a/Orco liposome (bottom) immobilisation followed by binding of the target ligand methyl salicylate (metsal). (B) Shows a close up view of the change in frequency with increasing concentrations of methyl salicylate (buffer with 1% DMSO, 1.6, 8, 20, 40 100, 200, 500 and 1000 μM) for the Or10a liposome (top) or Or10a/Orco liposome (bottom) immobilised QCM-D sensor. (C) Dose response curves showing detection of target ligand methyl salicylate, and control ligand methyl hexanoate (methex) when exposed to either Or10a liposomes or Or10a/Orco liposomes immobilized gold crystals. Error bars; standard deviation (SD) were generated using two repeats.

[0688] FIG. 12: Key steps involved in GFET fabrication on Si/SiO.sub.2 substrate, (a) Graphene coated Si/SiO.sub.2 substrate (b) etching the unwanted graphene layer (c) Graphene channel on Si/SiO.sub.2 substrate, (d) electrode deposition, and (e) electrode encapsulation.

[0689] FIG. 13: Schematic of liposome immobilization on graphene (a) functionalization of PBASE on graphene using π-π interaction (b) incubation of PBASE functionalised CNT with liposomes (c) tethering of liposomes with PBASE using nucleophilic substitution reaction.

[0690] FIG. 14: AFM images of (a) Or10a liposomes, (b) Or10a/Orco liposomes, (c) Or22a liposomes, (d) Or22a/Orco liposomes, (e) empty liposomes and, (f) Orco liposomes, immobilised on a graphene channel.

[0691] FIG. 15: GFET Device schematic and transfer characteristics. (a) Device schematic and circuit connections of a FET fabricated on a SiO.sub.2/Si substrate with encapsulated source and drain electrodes for liquid gate measurements. (V.sub.ds was chosen at 100 mV for CNT network FETs and 1 mV for GFETs). Transfer characteristic curves of the actual GFETs before (circle) and after (square) functionalisation of (b) Or10a, (c) Or10a/Orco, (d) Or22a, (e) Or22a/Orco, (f) Empty and (g) Orco liposomes (V.sub.ds was kept at 1 mV for all the measurements).

[0692] FIG. 16. Normalised real time sensing response of (a) Or10a and (b) Or22a, (c) Or10a+Orco and (d) Or22a+Orco liposome immobilised GFET sensors with the addition of increasing concentrations of target ligands (Or10a—methyl salicylate (MeSal), Or22a—methyl hexanoate (MeHex)).

[0693] FIG. 17. Normalized dose response curves for target ligands (Or10a—methyl salicylate (MeSal), Or22a—methyl hexanoate (MeHex)) and control ligands (Or10a and Or22a—E2-hexenal (E2Hex)) with (a) Or10a, (b) Or10a+Orco, (c) Or22a, and (d) Or22a+Orco liposomes. The lack of response from empty liposomes (a, c) and Orco only liposomes (b, d) to the target ligands are also shown. For both Or22a (e) AND Or10a (f) there can be seen a shift to the left for their respective dose-response curves when Orco is present in the liposomes.

[0694] FIG. 18. Overview of PMMA platform used for the bilayer sensor device. (A) 4-chamber design of array. (B) Schematic of droplet interface bilayer (DIB) formation between droplets (red and green) deposited on the base and manipulator electrode (blue) the original PMMA shape. The PMMA shape is shown as fully transparent to adequately display the position of the electrodes within the shape.

[0695] FIG. 19. Ion channel recordings from droplet interface bilayers (DIBs) containing (a) Or22a or (b) Or22a/Orco, and 10 mM methyl hexanoate, a known activator of Or22a. The DIB was formed between two aqueous droplets in a 1:1 undecane silicone oil mixture containing 1 mg/ml DPhPC, and the ion channel formation measured using the floating electrode set-up. The holding potential was varied as indicated. The reversal potential for Na.sup.+ is 46 mV for the above experiments. Data was sampled at 10 kHz and filtered at 0.8 kHz.

[0696] FIG. 20. Ion channel recording from a droplet interface bilayer (DIB) containing Or22a/Orco, and 10 μM methyl hexanoate. The DIB was formed between two aqueous droplets in a 1:1 undecane silicone oil mixture containing 1 mg/ml DPhPC, and the ion channel formation measured using the floating electrode set-up. The reversal potential for Na.sup.+ is 46 mV for this experiment and the holding potential was −100 mV. Data was sampled at 10 kHz and filtered at 0.8 kHz.

[0697] FIG. 21. (A) Ion channel recording from a droplet interface bilayer (DIB) containing Or71a, Orco, and 10 μM 4-ethyl guaiacol, a known Or71a agonist. The DIB was formed between two aqueous droplets in a 1:1 undecane silicone oil mixture containing 1 mg/ml DPhPC, as measured using the floating electrode set-up. The reversal potential for Na.sup.+ is −46 mV for this experiment. Data was sampled at 10 kHz and filtered at 0.8 kHz. (B) A second ion channel recording, as described in (A).

EXAMPLE 1—EXEMPLIFICATION OF THE SENSOR OF THE INVENTION WITH ELECTRICAL IMPEDANCE SPECTROSCOPY (EIS)

[0698] Previous unpublished data produced by the present inventors (resulting in the invention described in PCT/IB2017/058181) has shown that surprisingly an OrX can be used alone in an electronic sensor device, that is capable of detecting specific binding of analyte with significant improvement over insect OR-based sensor systems of the prior art.

[0699] The data of the present application shows that inclusion of Orco in addition to OrX surprisingly provides further significant improvements over both insect OR-based sensor systems of the prior art, and the OrX (alone)-based electronic sensor devices previously produced by the applicants forming the subject invention of PCT/IB2017/058181.

Summary

[0700] The applicants demonstrate the convenient, sensitive sensor device using insect OrX sequences. Two OrX receptors (Or10a, Or22a).sup.19 were each embedded on their own or with Orco in liposomes.sup.28 and functionalized on gold electrodes for EIS measurements under further optimized experimental conditions. Each of the OrX functionalized gold electrodes has shown a clear electronic response to its target ligands (Or10a to methyl salicylate, Or22a to methyl hexanoate).sup.20 starting at fM concentrations. The presence of Orco in the liposomes has an additive, or amplifying, effect on the OrX response, increasing the maximum response level and increasing the sensitivity of the OrX for its target ligand. The specificity of the binding is verified by testing each OrX liposomes and OrX/Orco liposomes functionalized electrode response to non-responding ligands. To further ensure the specificity the response of empty liposomes functionalized gold electrodes to the target ligands were also tested.

1.0 Experimental Methods

1.1 Materials

[0701] 6-mercaptohexanoic acid (MHA), N-hydroxysuccinimide (NHS), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide) (EDC), phosphate buffer saline (PBS) tablets, methyl salicylate, and methyl hexanoate were obtained from Sigma-Aldrich. 1.6 mm diameter gold (Au) disk electrode, coiled platinum (Pt) wire electrode and leakless silver/silver chloride (Ag/AgCl) electrode were purchased from BASi for electrochemical measurements.

1.2 Preparation of Purified OrX and Orco Subunits

[0702] The purification procedure is a variation on the one detailed in Carraher et al. 2013.sup.28. To his-tag affinity purify protein from baculovirus-infected Sf9 cells, 500 mL at 2×10.sup.6 mL.sup.−1 were infected with baculovirus at an MOI of 0.1, and incubated at 27° C. for 72 h. The cell pellet was collected by centrifugation at 3800 g for 10 min at room temperature and then resuspended in 40 mL of resuspension buffer A (20 mM Tris/HCl pH 7.5, 100 mM NaCl, 1× protease inhibitor cocktail (Roche Diagnostics GmbH, Germany)), with 25 U/mL Benzonase, then lysed by two passes on an Emulsiflex C5 emulsifier (Avestin, Germany) at 10,000-15,000 psi. The sample was then centrifuged at 1000 g for 5 min to remove whole cells and nuclei. The supernatant was removed and spun at 100,000 g for 1 h at 4° C. The membrane pellet was resuspended in 40 mL of buffer A with 1% w/v detergent (Fos-Choline 14 (FC14)) and rotated for 1 h at room temperature at 10 rpm. The sample was then centrifuged at 100,000 g for 1 h at 18° C. The supernatant was removed and loaded onto a 1 mL NiNTA column (GE Healthcare). The column was washed in ten column volumes of buffer B (20 mM Tris/HCl pH 7.5, 3.6 mM FC-14) with 300 mM NaCl and 20 mM imidazole, and a further ten column volumes of buffer B with 100 mM NaCl and 50 mM imidazole. Protein was eluted with four column volumes buffer B with 100 mM NaCl and 500 mM imidazole. Purity was assessed on Coomassie stained SDS-PAGE gels and Western blotting.

[0703] Purification was completed with a final size exclusion chromatography (SEC) step. The elution fractions from the NiNTA purification were pooled and centrifuged at 20,000 g for 5 min to remove aggregates and contaminants. Then 5 mL of sample was injected onto a Superdex 200 16/60 column (GE Healthcare) attached to an Akta-Pure chromatography system (GE Healthcare). The sample was run at 1 mL/min in buffer B with 100 mM NaCl, and 2 mL fractions were collected and concentrated using a 100 kDa MWCO Vivaspin2 filter unit (Sartorius, Goettingen Germany) and stored at −80° C.

1.3 Preparation of Liposome Associated OR Subunits

[0704] Liposomes were prepared using a phospholipid solution produced by evaporating solutions containing: phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylcholine (PC), and cholesterol (CH) at a molar ratio of 5:3:3:1 in a small glass tube under a stream of N2 gas, then desiccating under vacuum for 1 h.

[0705] These lipids were resuspended in 1 mL of rehydration buffer (10 mM HEPES pH 7.5, 300 mM NaCl) by vortexing for 5 min followed by sonicating on a Microson ultrasonic cell disrupter (Medisonic, USA) five times at 20% power for 10-20 s, placing the sample on ice between each sonication step for 1 min. To promote the formation of liposomes, 10 freeze/thaw steps were performed by transferring the tube from liquid nitrogen to a 40° C. water bath.

[0706] Liposomes were then sized by passing the lipid solution 11 times through a 100-nm polycarbonate membrane using an Avestin LiposoFAST extruder unit (Avestin, Germany). Glycerol was added at 10% of the final volume and aliquots at 10 mg/mL were snap frozen in liquid nitrogen and stored at −80° C.

[0707] Purified OrX and Orco subunits.sup.19 were reconstituted into the synthetic liposomes in a similar manner to the protocol of Geertsma et al. (2008).sup.34.

[0708] Prior to their use, liposomes were defrosted on ice and then destabilized by incubating with 0.2% CHAPS for 15 min at room temperature. Then 200 μg of purified odorant receptor.sup.28 was added to 1 mg of liposomes and rotated at 10 rpm for 1 h at room temperature. Excess detergent was removed by four additions of 25 mg of Bio-Beads SM-2 (Bio-Rad, USA) and incubation at 4° C. for 30 min, 2 h, overnight and a further 2 h respectively. The Bio-Beads were removed after each incubation period. The OrX or OrX/Orco integrated liposomes were pelleted by centrifugation at 100,000 g for 1 h, and were resuspended in 500 μL of rehydration buffer. Integration of OrXs and Orco into liposomes was assessed by density gradient ultracentrifugation (DGU) using Accudenz (Accurate Chemical & Scientific Corporation, USA). The integrated liposomes were brought to 40% Accudenz by the addition of an equal volume of 80% Accudenz solution, placed at the bottom of an ultra-centrifugation tube, and overlaid with 30% Accudenz solution, and DGU buffer (25 mM HEPES pH 7.5, 100 mM NaCl, 10% glycerol). The sample was then centrifuged at 100,000 g for 4 h at 4° C. Liposomes will float to the top of the gradient after Accudenz DGU due to their low density.

1.4 Electrode Preparation

[0709] Gold disk electrodes (1.6 mm diameter) were polished on alumina polishing pad with polishing alumina slurry for one minute for each electrode. The polished electrodes were rinsed with deionised water (Milli-Q, 18.2 MΩ cm) followed by ultrasonication in ethanol (LR grade) and deionised water until the residual alumina slurry was completely removed from the electrodes. Chronoamperometry at −1.4 V was applied onto all of the ultrasonicated electrodes to desorb the SAMs of the thiol present on the surface of the electrodes for 30 seconds using 0.1 M sodium hydroxide (NaOH) electrolyte solution in a three terminal electrochemical cell, Ag/AgCl (3 M NaCl, 0.209 V vs. SHE) reference electrode, coiled platinum wire as a counter electrode and gold disk as a working electrode, using a PalmSens3 potentiostat. Then, the electrodes were again rinsed with deionised water and ultrasonicated in ethanol and deionised water consecutively. Finally, cyclic voltammetry was performed for 10 cycles between −0.2 and 1.6 V, at a scan rate of 50 mV/s in 0.5 M sulphuric acid (H.sub.2SO.sub.4) solution to remove any other impurities (a three electrode cell, Ag/AgCl (in 3 M NaCl, 0.209 V vs. SHE) reference electrode, coiled platinum wire as a counter electrode and gold disk as a working electrode).

1.5 Self-Assembled Mono Layer (SAM) Preparation and Activation

[0710] 2 mM MHA was prepared by dissolving 1.36 μl of MHA in 5 ml ethanol (AR grade). The cleaned electrodes were immersed into MHA solution and incubated overnight. The next day, all the electrodes were washed with ethanol and deionised water thoroughly in order to remove the unreacted acid. A 2:1 mol:mol ratio of EDC:NHS (100 mM EDC, 50 mM NHS) was prepared in 2 ml PBS (pH=6.5) solution. Then, the electrodes were covered in 100 μl of this solution at 28° C. for an hour to activate the carboxylic (COOH) groups of the MHA.

1.6 OrX and OrX/Orco Associated Liposomes Immobilisation on Electrodes

[0711] PBS solution was prepared by immersing one tablet of PBS in 200 ml of milli-Q water (according to manufacturers instructions) and filtered using 0.2-μm syringe filter. The pH of the prepared buffer solution was measured with a pH meter. OrX liposomes or OrX/Orco liposomes were diluted 100 fold in PBS buffer solution (pH=7.4) and the COOH-activated electrodes were incubated in that buffer solution at room temperature for one hour. Then, the electrodes were washed extensively with PBS buffer solution to wash out any unbound liposomes.

1.7 Target Odorant Solution Preparation and Incubation

[0712] PBS (pH=7.4) was used as an electrolyte to conduct electrochemical measurements. PBS buffer was degassed for about 30 minutes prior to electrochemical measurements. Odorant solutions of concentration ranging from 1 aM to 1 μM were prepared by sequential dilution in PBS solution containing 1% DMSO. OR immobilized electrodes were incubated in relevant odorant solution for ˜30 minutes each and washed gently with PBS before EIS measurements.

1.8 Electrochemical Impedance Spectroscopy (EIS) Measurements

[0713] EIS measurements were done in a 3 electrode cell containing Ag/AgCl (3 M NaCl, 0.209 V vs. SHE) reference electrode, coiled platinum wire as a counter electrode and the gold disk as a working electrode at a fixed voltage of −0.7 using PalmSens potentiostat. Degassed PBS was used as an electrolyte.

2.0 Results

[0714] Insect olfactory receptors are comprised of OrX subunits in a complex with Orco subunits in cell membranes to produce an ion channel (FIG. 1).sup.17. In this study the authors investigate the effect of Orco on the ligand binding activity of OrXs embedded in liposomes. The experimental procedure consisting of the deposition of self-assembled monolayers (SAMs) of 6-mercaptohexanoic acid (MHA), activation of —COOH end groups with N-hydroxysuccinimide/1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide) (NHS/EDC) coupling, covalent attachment of ORs/liposomes and binding of the target odorant molecules as monitored by EIS, is presented in FIG. 2. The cleaned gold surface was incubated overnight with MHA to build SAMs with carboxylic acid end groups, as shown in the scheme. The —COOH end groups of the SAMs were further activated by exposing the gold surface to EDC/NHS solution to form N-hydroxysuccinimidyl ester, which can then link to the biomolecules. Finally, the electrode was incubated with OrX/liposome solutions, allowing the vesicles to covalently attach to the gold surface.

[0715] The authors used atomic force microscopy (AFM) to verify that the liposomes can be immobilised on to gold surfaces. FIG. 3 shows that a change can be seen in surface morphology and roughness profile from bare gold surface to the OR associated liposomes immobilized surface. The bare gold surface (FIG. 3 (a)) shows densely packed flat gold nanocrystals of various sizes with surface roughness value of around 2 nm. After SAM modification (FIG. 3 (b)) and NHS/EDC activation of SAM modified gold surface (FIG. 3 (c)), negligible changes in surface morphology were observed. When OrX liposomes were introduced to the EDC/NHS activated SAM modified gold surface (FIG. 3 (d)), the change in surface morphology was noticeable showing circular shaped liposomes immobilised on the surface. Variable sized round shaped liposomes were seen all over the surface in their native form i.e. no rupture or bilayer formation was observed which also indicates ORs are well retained in the membranes of liposomes. The large increase in surface roughness values (>30 nm) also demonstrates that ORs containing liposomes were successfully attached to the NHS/EDC activated SAM modified gold surface.

[0716] EIS measurements were performed on gold electrodes functionalised with OrX liposomes (either Or10a or Or22a), OrX/Orco liposomes, or empty liposomes prior to and after target ligand or control ligand incubation with increasing concentrations (1 aM to 100 μM). Dose response curves were obtained by defining sensor response as −(ΔR.sub.p/R.sup.o.sub.p) versus log[C(Ligand)]. FIG. 4a presents the EIS response in terms of Nyquist plot for the Or10a liposome functionalized sensors when exposed to a solution containing one of the known target ligands of Or10a, methyl salicylate, at various concentrations. The Nyquist plot showed decrease in EIS response after the addition of increasing concentration of methyl salicylate. The dose response curve (FIG. 4b) was obtained by plotting the change in polarization resistance −(ΔR.sub.p/R.sup.o.sub.p) versus log[conc(Ligand)], as the sensor response. Here, R.sub.p is the polarization resistance after Or10a liposome-target interaction and R.sup.o.sub.p is the polarization resistance before Or10a liposome-target interaction. The Or10a liposomes sensor responded to methyl salicylate with the limit of detection (LOD) of 0.1 pM and with negligible response to the control ligand methyl hexanoate control. The sensor functionalized with the empty liposomes showed negligible responses towards the positive and negative ligand suggesting OR is the key element to detect the odorants.

[0717] FIG. 5a presents the EIS response for the Or10a/Orco liposome functionalized sensors when exposed to its target ligand, methyl salicylate, at various concentrations. FIG. 5b compares the methyl salicylate dose response curve for Or10a/Orco liposomes with those obtained for Or10 liposomes, Orco liposomes and empty liposomes. The Or10a/Orco liposomes show a greater maximal response than Or10a liposomes, and also exhibit a greater sensitivity too, as reflected by the shift to the left of the dose response curve and a lower limit of detection of 1 fM. The Orco liposomes and empty liposomes both show a negligible response to methyl salicylate.

[0718] The authors investigate the effect of Orco on another example receptor, Or22a. FIG. 6a presents the EIS response for the Or22a liposome functionalized sensors when exposed to one of its target ligands, methyl hexanoate, at various concentrations. Again, the Nyquist plot showed decrease in EIS response after the addition of increasing concentration of methyl hexanoate. The dose response curve (FIG. 6b) shows that the Or22a liposomes sensor responded to methyl hexanoate with the limit of detection (LOD) of 1 fM and with negligible response to the control ligand methyl salicylate. The sensor functionalized with the empty liposomes showed negligible responses towards the positive and negative ligand suggesting the OR is the key element to detect the odorants.

[0719] FIG. 7a presents the EIS response for the Or22a/Orco liposome functionalized sensors when exposed to its target ligand, methyl hexanoate, at various concentrations. FIG. 7b compares the methyl hexanoate dose response curve for Or22a/Orco liposomes with those obtained for Or22a liposomes, Orco liposomes and empty liposomes. As expected, the Or22a/Orco liposomes show a greater maximal response than Or22a liposomes, and also exhibit a greater sensitivity too, as reflected again by the shift to the left of the dose response curve and a lower limit of detection of 0.1 fM. The Orco liposomes and empty liposomes both show a negligible response to methyl hexanoate.

3.0 Discussion

[0720] The applicants tested different Self-Assembled Mono layers (SAM layers) initially before identifying 6-mercaptohexanoic acid (MHA) as a linker of optimal length to bind the liposomes on to the gold electrode surface. Previously, 16-mercaptohexadecanoic (16-MHDA) acid was used to functionalize the gold surface and bind the liposomes onto the gold electrode. Results from that experiment did not show high sensitivity suggesting the liposomes were too far away from the electrode surface to give a detectable signal. To overcome that obstacle the applicants used the shorter 6-mercaptohexanoic acid instead. The applicants postulated that this linker being shorter would provide faster electron transfer between gold and liposomes, thus, any event occurring on the surface can be monitored in a more sensitive fashion. In the case of the two papers which immobilised mammalian odorant receptors in crude cell membranes, they used either 16-mercaptohexadecanoic acid (16-MHDA).sup.35 or 6-mecaptohexadecanoic acid (6-MHDA).sup.36 for SAM formation.

[0721] Comparative data shows that the insect OrX/Orco-EIS biosensor formats as disclosed here are more sensitive than both OrX-EIS biosensors, and other sensor formats that have been used with insect odorant receptors. Table 1 summarises the published data on odorant receptor based devices. The present device provides between 100-100,000-fold greater sensitivity than cell-based sensors.

TABLE-US-00001 TABLE 1 Comparison of insect odorant receptor sensor device data. Sensor/assay Receptor- Sensitivity approach analyte limit EC.sub.50 Ref Stable Sf21 BmOR1/Orco- 1 × 10.sup.−6 M 4.39 × 10.sup.−6 M 15 cell line on Bonnbykol microfluidics pheromone chip- BmOR3/Orco- 0.3 × 10.sup.−6 M 2.03 × 10.sup.−6 M fluorescence Bonnbykal pheromone Xenopus BmOR1/Orco- 10.sup.−8-10.sup.−6 M* 0.25 × 10.sup.−6 M 14 oocytes Bonnbykol on a pheromone microfluidics BmOR3/Orco- 10.sup.−8-10.sup.−6 M* 0.38 × 10.sup.−6 M device-two Bonnbykal electrode pheromone voltage clamping PxOR1/ 10.sup.−8-10.sup.−6 M* 2.52 × 10.sup.−6 M (TEVC) Orco-Z11- 16:Ald DOr85b/ 10.sup.−8-10.sup.−6 M* 45.6 × 10.sup.−6 M Orco to 2-heptanone Insect DmOr10a- ~10.sup.−10 M* ~10.sup.−10 M Present OrX-EIS methyl study device salicylate DmOr10a/ ~10.sup.−15 M* ~10.sup.−12 M Orco-methyl salicylate DmOr22a- ~10.sup.−15 M* ~10.sup.−12 M methyl hexanoate DmOr22a/ ~10.sup.−16 M* ~10.sup.−13 M Orco-methyl hexanoate *indicates value has been estimated from a visual assessment of dose response data plotted on a graph in the cited reference.

[0722] Table 2 summarises data obtained from cell assays. The present insect OrX-EIS sensor and OrX/Orco-EIS data is more sensitive than OrX/Orco expressed in HEK293 cells and Xenopus oocytes. Note in these systems some pheromone receptors (PRs) exhibit much lower sensitivity than normal odorant receptors, this is to be expected as these receptors are finely tuned to their pheromone target molecules.

TABLE-US-00002 TABLE 2 Overview of insect ORX/Orco cell assay data. Sensor/assay Receptor- Sensitivity approach Analyte limit Ec50 Ref Insect Sf9 EpOR1/ 10.sup.−14 M* 1.8 × 10.sup.−12 M 37 transient Orco-geraniol cell assay Insect Sf9 EpOR3/ 10.sup.−15 M* 1.1 × 10.sup.−13 M 37 transient Orco-Citral cell assay Insect Sf9 DmOr22a/ 10.sup.−12 M* 1.58 × 10.sup.−11 M 38 transient Orco-ethyl cell assay butyrate Insect Sf9 BmOr19/ 10.sup.−10 M* 4.69 × 10.sup.−9 M 5 transient Orco- cell assay linalool Insect Sf9 BmOr45/ 10.sup.−11 M* 1.44 × 10.sup.−10 M 5 transient Orco-benzoic cell assay acid Insect Sf9 BmOr47/ 10.sup.−14 M* 1.42 × 10.sup.−11 M 5 transient Orco-benzoic cell assay acid Insect Sf9 Am151/Orco- 10.sup.−10 M* 1.54 × 10.sup.−9 M 39 transient Floral mixture cell assay Insect Sf9 Am152/Orco- 10.sup.−10 M* 6.55 × 10.sup.−9 M 39 transient Floral mixture cell assay HEK293 EpOR3/Orco- 1.0 × 10.sup.−6 M 6 stable cell geranyl acetate assay HEK293 ApolOR1/Orco- 10.sup.−15 M* 10.sup.−13 M 40 stable cell (+ ApolPBP2, assay (E,Z)-6,11- hexadecadienal: pheromone) HEK293 HR13/Orco- 10.sup.−13 M 200 fM 41 stable cell PBP2 assay (+ pheromone) HEK293 HR13/Orco- 10.sup.−10 M 1.2 nM 41 stable cell DMSO assay (+ pheromone) HEK293 BmOR-1/ stable cell Orco-PBP 10.sup.−12 M 42 assay (+ pheromone) HEK293 DmOr22a/ Log = −7.5* Log = −6.38 43 stable cell Orco-methyl assay hexanoate HEK293 AgOr65/ Log = −7* Log = −6.54 43 stable cell Orco-eugenol assay HEK293 DmOr22a/ Log = −7* 1.17 × 10.sup.−6 M 44 stable cell Orco-methyl assay hexanoate) HEK293 AgOr48/ stable cell Orco-g- Log = −8* Log = −7.01 45 assay dodecalactone) Xenopus ECB (Z) OR3/ 10.sup.−9 M 12.5 × 10.sup.−9 M 2 oocytes Orco-E11 pheromone Xenopus ACB OR3/ 1 × 10.sup.−9 M 7 × 10.sup.−9 M 2 oocytes Orco-E12 pheromone Xenopus SexiOR13/ 3.158 × 10.sup.−6 M 46 oocytes Orco-Z9, E12-14:OAc pheromone. Xenopus SexiOR16/ 9.690 × 10.sup.−7 M 46 oocytes Orco-Z9- 14:OH pheromone. Xenopus OscaOR1/ 10.sup.−7 M* 10.sup.−6 M 47 oocytes Orco-E11- 14:OH pheromone. Xenopus MsiOR1/ 10.sup.−7 M* 10.sup.−6 M 48 oocytes Orco-Z11- 16:Ac pheromone Xenopus DiOR1/ 10.sup.−7 M* 10.sup.−6 M 48 oocytes Orco-Ell- 16:Ald pheromone Xenopus BmOrl/Orco- 34 × 10.sup.−6 M 49 oocytes bombykol pheromone Xenopus BmOrl/Orco- 5.9 × 10.sup.−6 M 50 oocytes bombykol pheromone Xenopus HVOR6/ 9.79 × 10.sup.−7 M 51 oocytes Orco-Z9- 14:ald pheromone Xenopus HVOR13/ 9.79 × 10.sup.−7 M 51 oocytes Orco-Z11- 16:ald pheromone Xenopus OnOrl/ 2.6 × 10.sup.−7 M 52 oocytes Orco-E12- 14:OAc pheromone Xenopus AgOR1/ 4.12 × 10.sup.−7 M 25 oocytes Orco-4- methylphenol Xenopus AgOR2/ 1.67 × 10.sup.−8 M 25 oocytes Orco-indole Xenopus AgOR8/Orco- 1.86 × 10.sup.−7 M 25 oocytes 1-octen-3-ol Xenopus AgOr10/ 1.37 × 10.sup.−7 M 25 oocytes Orco-indole Xenopus AgOr65/ 3.44 × 10.sup.−8 M 25 oocytes Orco-eugenol *indicates value has been estimated from a visual assessment of dose response data plotted on a graph.

4.0 Conclusion

[0723] This study has demonstrated the improved recognition ability of OrXs in the presence of Orco in olfactory biosensors based on electronic device platforms. OrXs embedded with the Orco subunit in liposomes which are functionalized on the gold electrodes show an increased sensitivity (below fM) and maximal response when compared with OrX liposomes. Compared with results from empty liposomes functionalized electrodes, no clear impedance response to target ligands are observed. The specific binding of each OrX has also been verified by testing the response to control ligands from the OrX liposome functionalized electrodes.

EXAMPLE 2—FURTHER EXEMPLIFICATION OF THE SENSOR OF THE INVENTION WITH ELECTRICAL IMPEDANCE SPECTROSCOPY (EIS)

Summary

[0724] The applicants further demonstrate the convenient, sensitive sensor device using an additional insect OrX sequence. Or35a.sup.19 was embedded on its own or with Orco in liposomes.sup.28 and functionalized on gold electrodes for EIS measurements in a similar manner to Example 1. As previously seen for the receptors Or10a and Or22a in Example 1, the presence of Orco in the liposomes has an additive, or amplifying, effect on the Or35a response, increasing the sensitivity of the OrX for its target ligand.

1. Experimental Methods

1.1 Materials

[0725] 6-mercaptohexanoic acid (MHA), N-hydroxysuccinimide (NHS), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide) (EDC), phosphate buffer saline (PBS) tablets, methyl salicylate, and methyl hexanoate were obtained from Sigma-Aldrich. 1.6 mm diameter gold (Au) disk electrode, coiled platinum (Pt) wire electrode and leakless silver/silver chloride (Ag/AgCl) electrode were purchased from BASi for electrochemical measurements.

1.2 Preparation of Purified Or35a and Orco Subunits

[0726] Or35a and Orco subunits were prepared as described in Example 1 section 1.2.

1.3 Preparation of OR Associated Liposomes

[0727] Or35a liposomes were prepared as described in Example 1 section 1.3 with the following alterations:

[0728] Prior to use, 1 mg liposomes (500 μl at 2 mg/ml) were defrosted on ice and then destabilized by incubating with 0.2% CHAPS for 15 min at room temperature. Then 50 μg of purified Or35a/Orco was added and rotated at 10 rpm for 1 h at room temperature. Excess detergent was removed by addition of 500 mg of Bio-Beads SM-2 (Bio-Rad, USA) and overnight incubation at 4° C. The tube was pierced at both ends and Or35a/Orco integrated liposomes were separated from the Bio-Beads by centrifugation at 5000 g for 1 min. All Or35a/Orco integrated liposomes samples were analysed by Western blot before being aliquoted and stored at −80° C. Integration of Or35a/Orco into liposomes was assessed by density gradient ultracentrifugation (DGU) using Accudenz (Accurate Chemical & Scientific Corporation, USA). The Or35a/Orco integrated liposomes were brought to 40% Accudenz by the addition of an equal volume of 80% Accudenz solution, placed at the bottom of an ultra-centrifugation tube, and overlaid with 30% Accudenz solution, and DGU buffer (25 mM HEPES pH 7.5, 100 mM NaCl, 10% glycerol). The sample was then centrifuged at 100,000 g for 4 h at 4° C. Liposomes floated to the top of the gradient after Accudenz DGU due to their low density.

2. Results

[0729] The applicants investigated the effect of Orco on the ligand binding activity of Or35a embedded in liposomes by performing EIS measurements as described in Experiment 1 section 2.0.

[0730] FIG. 8a presents the EIS response for the OR35a liposome functionalized sensors when exposed to one of its target ligands, E2-hexenal, at various concentrations. Again, the Nyquist plot showed decrease in EIS response after the addition of increasing concentration of E2 hexenal. The dose response curve (FIG. 8) shows that the Or35a liposomes sensor responded to E2 hexenal with the limit of detection (LOD) of 10 fM and with negligible response to the control ligand methyl salicylate. The sensor functionalized with the empty liposomes showed negligible responses towards the positive and control ligand suggesting the OR is the key element to detect the odorants.

[0731] FIG. 9a presents the EIS response for the OR35a/Orco liposome functionalized sensors when exposed to its target ligand, E2-hexenal, at various concentrations. FIG. 9b compares the E2-hexenal dose response curve for Or35a/Orco liposomes with those obtained for Or35a liposomes, Orco liposomes and empty liposomes. As expected, the Or35a/Orco liposomes show a greater maximal response than Or22a liposomes, and also exhibit a greater sensitivity too, as reflected again by the shift to the left of the dose response curve and a lower limit of detection of 0.1 fM. The Orco liposomes and empty liposomes both show a negligible response to methyl hexanoate.

[0732] FIG. 10 summarises clearly the shift in the dose response curves to the left for the OrX and OrX/Orco based EIS biosensors tested in Example 1 and 2. The dose response equation, EC.sub.50 and detection range for the OrX and OrX/Orco based EIS biosensors are summarised in Table 3. For the receptors tested in Example 1 and Example 2, the resulting sensors showed an improvement in sensor response in the presence of Orco in the form of lower LODs and EC.sub.50 values.

TABLE-US-00003 TABLE 3 Dose response equation, EC.sub.50 and detection ranges of OrX and OrX/Orco based EIS biosensors. Detection Receptor Analyte Dose Response Equation EC.sub.50 Range Or10a Methyl y = 0.31/(1 + exp 2.2 (±0.9) × 10.sup.−12 M 10.sup.−13 to 10.sup.−7 M salicylate (−0.80 × (x + 10.89))) Or10a/Orco Methyl y = −0.17 + (0.75)/(1 + 10.sup.∧ 7.39 × 10.sup.−14 M 10.sup.−15-10.sup.−7 M salicylate ((−14.13 − x) × 0.18)) Or22a Methyl y = 0.46/(1 + exp 1.1 (±2.1) × 10.sup.−12 M 10.sup.−15 to 10.sup.−5 M hexanoate (−0.53 × (x + 12.08))) Or22a/Orco Methyl y = −0.95 + (1.65)/(1 + 10.sup.∧ 1.46 × 10.sup.−4 M 10.sup.−16-10.sup.−5 M hexanoate ((−18.83 − x) × 0.08)) Or35a E2-hexenal y = 0.61*exp (−exp 2.8 (±2.3) × 10.sup.−13 M 10.sup.−14 to 10.sup.−6 M (−0.41*(x + 12.94))) Or35a/Orco E2-hexenal y = 0.11 + (0.72)/(1 + 10.sup.∧ 4.74 × 10.sup.−5 M 10.sup.−16-10.sup.−6 M ((−14.32 − x) × 0.26))

3. Conclusion

[0733] This study has demonstrated the improved recognition ability of Or35a in the presence of Orco in olfactory biosensors based on electronic device platforms. Or35a embedded with the Orco subunit in liposomes which are functionalized on the gold electrodes show an increased sensitivity (below fM) when compared with Or35a liposomes. Compared with results from empty liposomes functionalized electrodes, no clear impedance response to target ligands are observed. The specific binding of Or35a has also been verified by testing the response to a control ligand from the Or35a liposome functionalized electrodes.

EXAMPLE 3—EXEMPLIFICATION OF THE SENSOR OF THE INVENTION WITH QUARTZ CRYSTAL MICROBALANCE (QCM) PIEZOELECTRIC TRANSDUCER

Summary

[0734] The applicants have produced a convenient piezoelectric sensor device using the Drosophila melanogaster Or10a.sup.19 sequence embedded in liposomes in the absence and presence of the Orco sequence. Quartz Crystal microbalance with Dissipation monitoring (QCM-D) is a mass sensitive piezoelectric transducer, whose oscillation frequency changes with the mass loading on the crystal. The interaction between Or10a and the target ligand methyl salicylate was detected by monitoring the oscillation frequency changes of QCM-D sensor with Or10a liposomes and Or10a/Orco liposomes coupled to it. Or10a/Orco liposomes were found to have a greater response to methyl salicylate than Or10a liposomes. This result suggests the presence of the Orco subunit in the liposomes is having an additive effect on the response of an OrX amplifying its response to ligand binding. The specificity of the binding was verified by testing the response of Or10a liposomes and Or10a/Orco liposomes coupled to the QCM-D sensor to the control ligand methyl hexanoate, where there was negligible response.

1. Experimental Methods

1.1 Materials

[0735] 6-mercaptohexanoic acid (MHA), N-hydroxysuccinimide (NHS), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide) (EDC), phosphate buffer saline (PBS) tablets, and methyl salicylate were obtained from Sigma-Aldrich. Gold (100 nm) sensor crystals (QSX301) were obtained from ATA Scientific Instruments.

1.2 Preparation of OR Associated Liposomes

[0736] 1.2.1 Preparation of Purified OR Subunits

[0737] OR subunits were prepared as described in Example 1 section 1.2

1.2.2 Preparation of OR Associated Liposomes

[0738] Or22a liposomes were prepared as described in Example 1 section 1.3

1.3 Quartz Crystal Microbalance (QCM) Preparation and Data Collection

[0739] Gold (100 nm) sensor crystals were sonicated in ethanol and milli-Q water for 15 minutes each respectively. A 5:1:1 volume ratio of milli-Q water, ammonia (25%), and hydrogen peroxide (30%) was heated to 75° C. for 5 minutes and the sonicated crystals were placed in the heated solution for 5 minutes. Then the crystals were removed from the solution and rinsed with milli-Q water before drying with nitrogen gas. The clean gold crystals were thiol-functionalized by exposing them to 2 mM ethanolic solution of MHA overnight followed by washing with ethanol solution in order to remove excess or loosely bound molecules. NHS/EDC was prepared using 2:1 mol:mol ratio of EDC:NHS (100 mM EDC, 50 mM NHS) in 2 ml PBS (pH=6.5) solution. Each OrX/liposome stock solution was diluted 100 fold for QCM-D measurements in PBS buffer solution (pH=7.4). The SAM functionalized crystals were then placed into the Q-sense analyser instrument (Biolin Scientific) chamber and flowed with the NHS/EDC, Or10a/liposomes or Or10a/Orco liposomes, and various concentrations of methyl hexanoate (1.6 μM, 8 μM, 20 μM, 40 μM, 100 μM, 200 μM, 500 μM and 1000 μM) in PBS buffer solution containing 1% DMSO to measure the changes in frequency (Δf) and dissipation (ΔD) values.

2. Results

[0740] FIG. 11 (a) shows the change in frequency upon the SAM and NHS/EDC modification, followed by Or10a liposome or Or10a/Orco liposome immobilisation on the quartz crystal and then binding of the target ligand methyl salicylate. When a binding event occurs on the crystal this results in an increase in the mass reducing the frequency of oscillation.sup.53. Thus the mass of the sensor increases with SAM, NHS/EDC, and Or10a liposome or Or10a/Orco immobilisation. However, in the case of methyl salicylate binding an increase in the frequency is observed for both types of liposomes (FIG. 11 (b)). Without wishing to be bound by theory, the inventors suggest this loss of mass on the sensor is due to the binding of methyl salicylate to the Or10a receptor causing a release of water and ions from inside the Or10a liposomes i.e. the Or10a is forming a functional ion channel. Likewise, the binding of methyl salicylate to the Or10a receptor causing a release of water and ions from inside the Or10a/Orco liposomes i.e. Or10a/Orco is forming a functional ion channel. However, the Or10a/Orco liposomes exhibit a much larger change in frequency, indicating the Orco subunit is amplifying the response from the Or10 a subunit. Comparison of dose response curves for both types of liposomes shows the Or10a/Orco liposomes respond more strongly at higher methyl salicylate concentrations (FIG. 11c). In both cases, this increase in frequency occurs with increasing concentrations of methyl hexanoate between 1.6 to 1000 μM indicating that methyl salicylate is binding specifically to the Or10a receptor, as this increase in frequency is not observed with the control ligand methyl hexanoate (FIG. 11c). Detection of ligand binding at the μM level equivalent to parts-per-trillion (ppt) concentration is on par with what has been seen with C. elegans ODR-10.sup.54.

3. Conclusion

[0741] This study has demonstrated the recognition ability of OrXs in olfactory biosensors based on electronic device platforms. OrXs in liposomes which are functionalized on quartz crystal microbalance (QCM) piezoelectric sensors can specifically detect their target ligand. An OrX in combination with Orco shows a stronger response to their target ligand, indicating Orco has an additive or amplifying effect on the response of an OrX to its ligand. The response is OrX specific as no clear piezoelectric response was observed to the control ligand. OrX/Orco liposomes functionalized QCMs show great promise to specifically and sensitively detect their target ligands.

EXAMPLE 4—EXEMPLIFICATION OF THE SENSOR WITH GRAPHENE FIELD EFFECT TRANSISTORS (GFETS)

Summary

[0742] The applicants have produced a convenient GFET sensor device using the Drosophila melanogaster Or10a and Or22a sequences.sup.19 embedded in liposomes in the absence and presence of the Orco sequence. The experimental results showed the in vitro sensing of insect ORs with GFET platforms. Each of the OrX functionalized GFETs has shown a clear electronic response to its target ligands (Or10a to methyl salicylate, Or22a to methyl hexanoate).sup.2° starting at pM concentrations. The presence of Orco in the liposomes has an additive, or amplifying, effect on the OrX response, increasing the sensitivity of the OrX for its target ligand down to fM concentrations. The specificity of the binding is verified by testing each OrX liposomes and OrX/Orco liposomes functionalized GFET response to non-responding ligands. To further ensure the specificity the response of empty liposomes functionalized GFETs to the target ligands were also tested.

1. Experimental Methods

1.1 Materials

[0743] The nitrogen 99.99%) and oxygen (99.7%) for the experiments were purchased from BOC limited New Zealand. Deionised (DI) water (18.2 MΩ) used was obtained from a Sartorius (Arium® 611 VF) DI water plant. For GFET fabrication a wafer containing mechanically transferred CVD Graphene on 300 nm SiO.sub.2/p-type Si substrate was purchased from Advanced Chemical Supplier, CA, USA; the positive photoresist AZ1518 was purchased from Microchem, Germany; and 1-Pyrenebutyric acid N-hydroxysuccinimide ester (PBASE) (95%, Sigma Aldrich) was used as the molecular linker to tether the OrX and OrX/Orco liposomes to the surface of the graphene present on the GFET device.

1.2 Preparation of OR Associated Liposomes

1.2.1 Preparation of Purified OR Subunits

[0744] OR subunits were prepared as described in Example 1 section 1.2

1. 1.2.2 Preparation of OR Associated Liposomes

[0745] OR liposomes were prepared as described in Example 1 section 1.3

1.3 Preparation of GFET Sensors

[0746] The GFETs used in this study were fabricated from a predeposited graphene film on 300 nm SiO.sub.2 coated Si substrate from ACS Suppliers, USA. The FETs consist of a channel with dimensions of 100 μm width and 40 μm length. The key steps involved in fabricating the GFET on a Si/SiO.sub.2 substrate is schematically illustrated in FIG. 12. The source and drain electrodes were defined by photolithography and formed by successive deposition of Cr and Au. Both the drain and source electrodes were then encapsulated using AZ1518 photoresist and a channel with dimensions of 100 μm width and 10 μm length was left open to the environment for gating and functionalisation.

[0747] To fabricate GFETs, a wafer containing mechanically transferred CVD Graphene on 300 nm SiO.sub.2/p-type Si substrate was purchased from Advanced Chemical Supplier, CA, USA. The wafer was first cleaved into squares chips with a dimension of 12 mm×12 mm. The chips were rinsed in acetone and IPA to remove the contaminants on the graphene surface. Then the alignment marker deposition was carried out by thermal evaporation using an Angstrom engineering—Nex Dep 200 evaporator. The devices were mounted on a rotating stage and the chromium and gold metal sources were loaded. Chrome plated tungsten rods (Kurt J. Lesker Company) were used as the chromium source. Pieces of gold wire (99.99%, Kurt J. Lesker Company) were loaded into a tungsten boat (Kurt J. Lesker Company) and loaded into the evaporation chamber. The chamber was evacuated to 2×10.sup.−6 mTorr and 5 nm of chrome and 50 nm of gold was evaporated successively. The chamber was cooled down and vented with nitrogen. The lift-off was carried out by soaking the devices in acetone for 10 min and then washing in IPA before being dried with nitrogen. The channel area was defined using AZ1518 photoresist and the graphene film on the rest of the chip was etched using 200 W oxygen plasma at 600 mTorr for 1 min using a reactive ion etcher (Oxford instruments, Plasmalab 80 Plus). The top contacts were then deposited by successive thermal evaporation of 5 nm Cr and 50 nm Au after defining them by photolithography. The electrodes were encapsulated by AZ 1518 photoresist. The encapsulated graphene FETs were cleaned under 50 W oxygen plasma for one min at 200 mTorr pressure and 20 SCCM oxygen flow to remove the residual photoresist on the graphene channel. Then the devices were hard-baked at 200° C. for 10 min on a hotplate and washed in acetone and IPA before functionalisation.

1.4 Functionalisation of OrX and OrX/Orco Liposomes on GFETs

[0748] The OR liposomes were functionalised onto the graphene surface via a non-covalent route using PBASE as the molecular linker as shown in FIG. 13. The OR liposomes were diluted at 1:10 ratio with 1×PBS (pH7.4). The cleaned GFETs were immersed in 1 mM PBASE solution in methanol for 1 hour. The devices were washed three times in methanol and subsequently washed three times in 1×PBS to remove the excess PBASE and residual methanol in the channel respectively. The OR liposomes were diluted 1:10 with 1×PBS (pH 7.4) and 100 μL of the OR dilution was placed in the graphene channel and incubated at room temperature for one hour in a closed petri dish. After OR liposome functionalization, the devices were washed in 1×PBS for 10 s before measurements.

[0749] The attachment of OR liposomes to the graphene surface was verified by AFM using a tabletop AFM (Nanosurf, NaioAFM). Imaging was carried out using tapping mode with dynamic applied force. AFM images after OR liposome functionalisation were carried out in air. The functionalised graphene FETs were washed in DI water and the excess water was drained. The functionalised devices were dried under a Nitrogen stream before imaging. Gwydion (V. 2.47) and SPIP software packages were used to analyse the AFM images. FIG. 14 shows Or10a, Or10a/Orco, Or22a, Or22a/Orco, empty liposomes and Orco liposomes immobilised on the graphene surface. The average size of the liposomes used in this study was estimated as 128±43 nm. The AFM images confirmed that the spherical structure of the liposomes was preserved after functionalisation.

1.4 Electrical Characterisation of OR Liposome GFETs

[0750] Electrical sensor measurements of OR liposome functionalised GFETs were carried out using top liquid gate morphology as shown in the schematic in FIG. 15 (a). A PDMS well was used to constrain the electrolyte to the channel region. Devices were electrically characterized using an Agilent 4156C parameter analyser and a Rucker and Kolls probe station with micromanipulators. Ag/AgCl standard electrode was used as the gate electrode for liquid gate measurements. The transfer characteristics of the different graphene FETs (FIG. 15 (b) to (g)) were measured at V.sub.ds=1 mV while the liquid gate voltage V.sub.lg was swept from −0.5 V to 1 V with an interval of 20 mV.

1.5 OR Liposome GFET Sensor Measurements

[0751] The OR liposome immobilised GFET device with a PDMS well mounted on it was placed onto the probe station and the source and drain connections were made by micromanipulators. 100 μl of PBS containing 1% dimethyl sulfoxide (DMSO) was added to the well and the Ag/AgCl standard electrode was placed into the buffer. The stock solution of ligands at 100 mM concentrations were prepared by dissolving them in DMSO as they are not stable in aqueous buffer. The stock solution was stored at 4° C. The ligand solution for sensing was prepared by diluting the stock solution in 1×PBS buffer containing 1% DMSO to set concentrations from 10 fM to 100 pM. The ligand solution was added to the PDMS well at three-minute intervals to make final concentrations from 1 fM to 10 pM. The real-time sensor measurement was carried out by continuously measuring the I.sub.ds at an interval of 1 s. The gate voltage V.sub.lg was maintained at 0 V throughout the measurement via the Ag/AgCl reference electrode.

2. Results

[0752] The sensing performance of OR liposome functionalised GFET sensors was tested. OR liposomes with and without the co-receptor Orco were used for sensing tests. Four sets of sensors were fabricated by immobilising Or10a, Or10a/Orco, Or22a and Or22a/Orco liposomes and tested.

[0753] Or10a and Or10a/Orco liposome sensors were tested against their positive ligand, methyl salicylate. Or22a and Or22a/Orco liposome sensors were tested against their positive ligand, methyl hexanoate. These sensors were also tested using E2-hexenal as a ligand control. The response of empty liposome and Orco liposome functionalised GFET sensors were tested against both methyl salicylate and methyl hexanoate as controls. Each experiment was carried out in triplicate to reduce the experimental and measurement errors.

[0754] FIG. 16 shows the normalised real time sensing response of all four sensors with the addition of increasing concentrations of their positive ligands. It shows that each of the liposome based sensors with and without Orco produced a dose dependent response specific to the positive ligand and not to the control ligand. Neither empty liposomes nor Orco containing liposomes respond to the positive ligands confirming selective binding is due to the presence of the OrX. FIG. 17 summarises the effect of the presence of Orco on the response of each OrX to its positive ligand. The presence of Orco results in an increase in sensitivity as demonstrated by the shift of their dose response curves to the left and the lowering of their LODs from pM to 100 fM levels. Table 4 summarises the Ec50 and detection range for the OrX and OrX/Orco combinations.

TABLE-US-00004 TABLE 4 Dose response equation, EC.sub.50 and detection ranges of OrX and OrX/Orco based GFET biosensors. Detection Receptor Analyte Dose Response Equation EC.sub.50 Range Or10a Methyl y = 0.138 × 4.62 × 10.sup.6 × x.sup.1-0.38/ 15.56 × 10.sup.−12 M 10.sup.−12 to 10.sup.−8 M salicylate 1 +4.62 × 10.sup.6 × x.sup.1-0.38 Or10a/Orco Methyl y = 0.129 × 1.45 × 10.sup.5 × x.sup.1-0.56/  2.07 × 10.sup.−12 M 10.sup.−13-10.sup.−8 M salicylate 1 +1.45 × 10.sup.5 × x.sup.1-0.56 Or22a Methyl y = 0.12 × 2.73 × 10.sup.6 × x.sup.1-0.41/ 12.09 × 10.sup.−12 M 10.sup.−12 to 10.sup.−8 M hexanoate 1 +2.73 × 10.sup.6 × x.sup.1-0.41 Or22a/Orco Methyl y = 0.131 × 4.02 × 10.sup.6 × x.sup.1-0.5/  3.91 × 10.sup.−12 M 10.sup.−13-10.sup.−8 M hexanoate 1 +4.02 × 10.sup.6 × x.sup.1-0.5

3. Conclusions

[0755] This study has demonstrated the improved sensitivity of both Or10 and Or22a in the presence of Orco in olfactory biosensors based on GFET devices. Both, Or10a and Or22a embedded with the Orco subunit in liposomes which are functionalized on graphene show an increased sensitivity (fM) when compared with the receptors on their own in liposomes. Compared with results from empty liposomes and Orco liposomes functionalized graphene, no clear electronic response to target ligands are observed. This confirms that although Orco is not directly involved in ligand binding, its presence in the liposomes further attenuates the sensitivity of the OrX to its target ligand.

EXAMPLE 5—EXEMPLIFICATION OF THE BILAYER SENSOR DEVICE WITH DROPLET INTERFACE BILAYERS (DIBS)

Summary

[0756] The applicants have shown that functional ionotropic olfactory receptors (ORs) have been incorporated into proteoliposomes and fused with artificial bilayers where the reversible binding of an odorant can be measured electrically.

1. Experimental Methods

1.1 Materials

[0757] Phosphate buffer saline (PBS) tablets, methyl salicylate, and methyl hexanoate were obtained from Sigma-Aldrich. All lipids were sourced from Avanti polar lipids. All other chemicals were purchased from Merck, UK, unless otherwise specified. Double-distilled ‘ultrapure’ water (Millipore, Milli-Q:18.2 MΩ cm) was used throughout.

1.2 Preparation of Purified OrX and Orco Subunits

[0758] OrX and Orco subunits were prepared as described in Example 1 section 1.2 with the following alteration:

[0759] Following the resuspension of the membrane pellet, the sample was centrifuged at 100,000 g for 1 h at 4° C. rather than 18° C.

1.3 Preparation of OR Associated Liposomes

[0760] OR associated liposomes were prepared as described in Example 1 section 1.3 with the following alteration:

[0761] The incubation step with Bio-Beads was performed overnight.

1.4 Preparation of Lipid Droplets

[0762] DPhPC (Avanti, 4ME 16:0 PC) was dissolved in chloroform and aliquoted before dried under a stream of nitrogen to form a thin lipid film. This was then placed in a desiccator under vacuum for 14 hours Aliquots were then stored under argon at <−20° C. Before use, undecane (Merck, UK) was added to dissolve the lipid to make a solution of 10 mg/ml. This was diluted in AR20 silicone oil (Merck, UK) and undecane (to make a final AR20 silicone oil undecane ratio 1:1) to make a 1 mg/ml solution. Undecane and AR20 silicone oil was prefiltered using a 0.22 μm filter before use.

1.5 Electrode Preparation

[0763] Silver electrodes (0.5 mm diameter, >99% purity, Merck) were cut to appropriate lengths and prepared with fine grit sandpaper before incubated in sodium hypochlorite solution (Fluka, UK) for 1 hour. The electrodes were then washed in ddH.sub.2O before inserted into the electrophysiology array wells or affixed to the manipulator.

1.6 Electrophysiology Array

[0764] A multiwell array made from poly(methyl methacrylate) (PMMA) was designed using computer assisted design software (FreeCAD, https://www.freecadweb.org/) and milled using a subtractive computerized numerical control (CNC) machine (Roland Modela MDX-40A). FIG. 18 shows an overview of this PMMA platform. (A) shows the 4-chamber design of array. (B) shows a schematic of DIB formation between droplets (red and green) deposited on the base and manipulator electrode (blue) the original PMMA shape. The PMMA shape is shown as fully transparent to adequately display the position of the electrodes within the shape.

1.7 Electrophysiology Setup

[0765] The electrophysiological recordings were taken using Picot (Tecella, USA) amplifier within a faraday cage containing.

1.8 Droplet Interface Bilayer Formation

[0766] Bilayers were formed between two droplets formed within a PMMA chamber filled with 1 mg/ml DPhPC in undecane and AR20 silicone oil (1:1 ratio). The first step in this process was to deposit a 50 nl droplet onto the stationary silver electrode positioned at the base of the well; this droplet consisted of an aqueous solution containing 300 mM NaCl, 10 mM HEPES (pH 7.4), containing the odorant receptor proteins in proteoliposomes (at a 1:20 dilution from the proteoliposome preparation), and 0.1-1 μM of the odorant. The second droplet was mounted onto a second silver electrode which was held in the oil and lipid mixture using a YOU-3 manipulator (Narishige, Japan). The second droplet had a total volume of 50 nl containing 50 mM NaCl and 10 mM HEPES (pH 7.4). Both droplets were deposited using a 0.5 μl syringe (Hamilton, USA). To ensure that a stable phospholipid monolayer had formed around each droplet, the droplets were left on the electrodes for 5 min before being gently brought together using the YOU-3 manipulator. Once the droplets were in contact a bilayer formed spontaneously within 1 minute (as determined by visual assessment and an increase in the bilayer capacitance as measured with a capacitance voltage protocol using the Pico2 amplifier). Insertion of an active channel was determined by a change in current while clamping the voltage at 50 mV. This process took up to 45 min. If no insertion was seen at this point the experiment was discarded. Droplet interface bilayer experiments were conducted at 22.0±1.5° C.

1.9 Recording Parameters

[0767] Currents were recorded with a Pico2 or an eONE-HS amplifier (Tecella, USA and Elements, Italy, respectively) with built-in digitizers, operating in gap-free acquisition mode at a sampling frequency of 20 kHz, and using a 0.8 or 1.5 kHz low pass filter. All experiments were conducted using the voltage-clamp approach. The voltage across the membrane was clamped at various potentials, ranging from −200 mV to 200 mV.

1.10 Manual Data Analysis

[0768] Data were analysed using ANA (Dr Pusch, Genoa), EDR (Elements, Italy) and WinWCP (Dr Dempster, University of Strathclyde). Single channel currents were measured as step increases in the amplitude of the current observed. When measuring the current at different holding potentials the baseline current might be at a different level. To compensate for this, the change in current is given in relation to the baseline, as is common practice. At holding potentials below the electrochemical equilibrium value, the channel openings are shown as downward deflections. Inversely, channel openings are shown as upward deflections from the baseline when the holding potential is greater than the reversal potential.

2. Results

[0769] FIG. 19 shows ion channel recordings from droplet interface bilayers (DIBs) formed between two aqueous droplets in a 1:1 undecane silicone oil mixture containing 1 mg/ml DPhPC, as measured using a floating electrode set-up (see FIG. 18). Proteoliposomes containing Or22a only (FIG. 19a) and Or22a/Orco (FIG. 19b) were fused with the DIBs to allow insertion of the receptor subunits. The solutions used contains 10 mM HEPES (pH 7.4 with NaOH), either 300 mM NaCl or 50 mM NaCl, supplemented with 10 μM Methyl hexanoate, a known Or22a agonist. For the Or22a only DIB experiment (FIG. 19a) the holding potential was varied between +/−150 mV, +/−100 mV, and +/−50 mV. No ion channel activity is observed at any holding potential indicating that Or22a cannot form an active ion channel on its own. For the Or22a/Orco DIB experiment (FIG. 19b) the holding potential was varied between +/−100 mV, +/−50 mV and +/−25 mV. In this case ion channel activity is observed at +/−100 mV, +/−50 mV and −25 mV indicating that Or22a can form an active ligand gated ion channel when combined with Orco. FIG. 20 shows a second independent Or22a/Orco DIB experiment acquired at a holding potential of −100 mV. In this case multiple open states can be identified, likely due to the insertion of multiple Or22a/Orco channel complexes.

[0770] FIG. 21 shows two ion channel recordings from droplet interface bilayers (DIBs) formed between two aqueous droplets in a 1:1 undecane silicone oil mixture containing 1 mg/ml DPhPC, as measured using a floating electrode set-up (see FIG. 18) at a holding potential of 0 mV (FIG. 21a) and −100 mV (FIG. 21b). Proteoliposomes containing Orco/Or71a were fused with the DIB to allow insertion of the receptor subunits. The solutions used contains 10 mM HEPES (pH 7.4 with NaOH), either 300 mM NaCl or 50 mM NaCl, supplemented with 10 μM of the target ligand 4-ethyl guaiacol. Both DIB experiments show that Or71a in the presence of Orco can form an active ligand gated ion channel.

3. Conclusions

[0771] This study has further confirmed that the presence of Orco affects the sensitivity of OrXs in olfactory biosensors based on electronic device platforms. In this case, when two OrXs (Or22a and Or71a) are inserted independently with the Orco subunit into lipid bilayers both OrX/Orco complexes exhibit ion channel activity in the presence of a target ligand for the OrX. However in the absence of Orco this ligand gated activity is not exhibited highlighting the role of Orco in forming an active ion channel. This data demonstrates the potential to use OrX/Orco complexes in lipid bilayer based sensor devices to detect specific volatile organic compounds based on their ion channel activity response.

EXAMPLE 6—EXEMPLIFICATION OF THE SENSOR WITH SURFACE PLASMON RESONANCE IMAGING

Summary

[0772] The applicants describe a convenient SPRi sensor device using insect odorant receptor (OrX) subunits embedded in the membrane mimic including liposomes and nanodiscs in the absence and presence of the Orco sequence. Each of the OrX functionalized SPR sensors show a clear electronic response to its target ligand. The presence of Orco in the membrane mimic has an additive, or amplifying, effect on the OrX response, increasing the sensitivity of the OrX for its target ligand. The specificity of the binding is verified by testing each OrX and OrX/Orco functionalized SPRi sensors response to non-responding ligands.

1. Experimental Methods

1.2 Preparation of OR Associated Liposomes and Nanodiscs

[0773] 1.2.1 Preparation of Purified OR Subunits

[0774] OrX and Orco subunits are prepared as described in Example 1 section 1.2. OrX and Orco subunits have a Cysteine residue engineered at their N termini to enable their direct coupling to the gold surface of an SPRi prism.

[0775] 1.2.2 Preparation of OR Associated Liposomes

[0776] OrX and Orco liposomes are prepared as described in Example 1 section 1.3

[0777] 1.2.3 Preparation of OR Associated Nanodiscs

[0778] Nanodiscs are prepared using a protocol modified from Bayburt et al. 2010 and 2003.sup.55, 56. Nanodiscs were formed at an MSP:protein:lipid ratio of 1:0.2:150. The required amount of lipid is removed from the 100 mg/mL stock and dried under a constant stream of nitrogen gas, then further dried under vacuum overnight. The lipids are resuspended in the required volume of buffer (20 mM Tris/HCl pH 7.5, 100 mM NaCl, 50 mM sodium cholate) and sonicated, resulting in a clear lipid stock at 20 mg/mL concentration. Purified odorant receptor protein in detergent buffer is mixed with the MSP1E3D1 and POPC lipid at the required ratio and incubated on ice for 1 hour. To initiate the reconstitution by removing detergents from the system, Bio-beads SM2 (Bio-Rad #1523920) are added to the sample at a 1:1 weight:volume ratio and the mixture is incubated at 4° C. overnight with constant rotation. Bio-beads are then removed and the incorporated nanodiscs are frozen at −80° C. until required.

1.3 Preparation of OrX and OrX/Orco SPRi Sensors

[0779] OrX and OrX/Orco in liposomes or nanodiscs are immobilised as defined spots onto the gold surface of an SPRi prism according to the protocol used by Hurot et al. 2019 for the immobilisation of vertebrate odorant binding proteins (OBPs).sup.57. OrX and OrX/Orco complexes are immobilised directly via the N-terminal Cysteine residue. Immobilisation occurs at an appropriate density to ensure a self-assembly of liposomes or nanodiscs on the gold surface that yields a liposome or nanodisc monolayer. This prevents the formation of additional disordered layers of liposomes or nanodiscs which prevent target ligands accessing the binding pockets of OrXs or OrX/Orco complexes directly attached to the gold layer. A monolayer that completely covers the gold surface is obtained to block non-specific binding of target ligands to the gold surface.

1.4 Detection and Analysis of Ligand Binding by OrX and OrX/Orco SPRi Sensors

[0780] The binding of VOCs to OrX and OrX/Orco liposomes or nanodiscs are detected using an appropriate SPRi apparatus and analysed as described by Hurot et al. 2019.sup.57. The OrX or OrX/Orco immobilised gold surface is exposed to different concentrations (fM to nM) of target ligand or control ligand. Ligand binding is measured as a change reflectivity as compared to the baseline prior to addition of the ligand. Each experiment is carried out in triplicate to reduce the experimental and measurement errors.

2. Results

[0781] The sensing performance of OrX and OrX/Orco liposome or nanodisc functionalised SPRi sensors are tested against a positive ligand specific to the OrX subunit and a control ligand to which the OrX should not bind. The response of empty liposome and Orco liposomes, or empty nanodiscs and Orco nanodiscs functionalised SPRi sensors are also tested against the positive ligand.

[0782] In the case of both membrane display formats, the OrX subunit on its own binds sensitively to the positive ligand producing a dose response curve, but does not respond to the control ligand. When the Orco subunit is also present, the presence of Orco is expected to result in an increase in sensitivity as demonstrated by the shift of their dose response curves to the left and the lowering of their LODs. Neither empty liposomes nor Orco containing liposomes respond to the positive ligands confirming selective binding is due to the presence of the OrX.

3. Conclusions

[0783] This study is expected to demonstrate the improved sensitivity of OrXs in the presence of Orco in olfactory biosensors based on SPRi devices. It is believed that OrXs embedded with the Orco subunit in liposomes or nanodiscs which are functionalized on the gold surface of an SPRi glass prism will show an increased sensitivity when compared with the OrX receptors on their own. Compared with results from empty liposomes or nanodiscs and Orco containing liposomes or nanodiscs, respectively, no clear electronic response to target ligands are observed. This is expected to confirm that although Orco is not directly involved in ligand binding, its presence in the liposomes further attenuates the sensitivity of the OrX to its target ligand.

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