Patent Description:
A semiochemical is a compound that is secreted by organisms, which modifies the behaviour and/or development of another organism. Semiochemicals are categorised into intraspecific semiochemicals, pheromones, a compound or group of compounds that are released by an organism and induce a response in an individual of the same species, and interspecific semiochemicals, allelochemicals, which stimulate organisms of different species. Pheromones are critical for communication between insects of the same species; these may include sex pheromones, aggregation pheromones, and alarm pheromones.

Pheromones can be further categorised into releasers, pheromones which induces an immediate behavioural change, and primers, pheromones which initiate a complex set of physiological or developmental changes, but may result in no immediate behavioural change.

Semiochemicals are used in host location, mating and enemy warning systems. Although semiochemicals are widely employed by insects for communication, communication chemistry and potential semiochemicals have been identified in many other organisms including mammals, birds and fish.

Aphid species, including the pea aphid, Acyrthosiphon pisum, have been shown to employ sex pheromones (e.g. (4aS,<NUM>S,7aR)-nepetalactone and (<NUM>R,<NUM>aS,<NUM>S,<NUM>aR)-nepetalactol) and an alarm pheromone, in addition to a range of allelochemistry generally utilised for host-location.

Semiochemicals have the potential to be used in pest management, by using mating disruption, pheromone traps, push-pull strategies and recruitment of natural enemies. Synthetic sex pheromone components have been used to catch male aphids and recruit foraging parasitic wasps, Aphidius ervi Haliday and Praon barbatum Mackauer, in the field. The synthetic sex pheromone components have also been found to attract other aphid natural predators, such as lacewings (Chrysopa cognata).

The alarm pheromone (E)-β-farnesene (EBF) has been shown to be repellent to aphids in behavioural studies, whilst attractive to natural enemy predators and parasitoids. A hexaploid commercial variety of wheat, Triticum aestivum cv. Cadenza (Poaceae), has been genetically engineered to biosynthesise and release EBF. Evaluation in field trials showed the transformed wheat variety was not significantly different from non-transformed varieties in managing aphids (assessed by aphid numbers and number of parasitized aphids), although controlled environment studies had demonstrated its effectiveness. This is likely due to the release rate of the alarm pheromone from the plant being consistent and steady, in contrast to a natural quick burst of pheromone produced by aphids.

Plant-derived semiochemicals also have practical applications; (Z)-jasmone has been found to be effective in reducing aphid numbers, attracting parasitic wasps and inducing the production of repellent volatiles in crops such as wheat, cotton and sweet peppers. (S)-Germacrene D has been identified as a potent repellent for aphids, but has little potential for commercial application in crop protection due to its chemical instability and cost of production. Development of more stable analogues which have comparable behavioural activity could be developed, particularly using modified terpene synthases and unnatural substrates.

Further advancement of the use of semiochemicals in pest management could stem from a deeper understanding of molecular recognition processes in the olfactory systems of pests. This may lead to 'ab initio' design of ligands, which may have similar behavioural effects as other olfactory ligands, but better prospects for commercial producibility. Currently, understanding of the olfactory system is limited, though two major groups of proteins are involved - olfactory receptors (ORs) and odorant binding proteins (OBPs) - both of which could provide potential pest management targets.

Given the significant value in understanding olfactory systems and semiochemicals in various industries, such as the food, health and pharmaceutical industries, there exists a need to develop new semiochemicals and sensors. The present invention addresses this need.

<NPL>, describe analogues of (E)-β-Farnesene that bind to odorant-binding proteins of the pea aphid Acyrthosiphon pisum. de <NPL>, describe relations between Acyrthosiphon pisum odorant-binding protein expression patterns and their functional implications.

<NPL>, review odorant-binding proteins and other soluble proteins as detecting elements in biosensors for odours.

<NPL>, review binding protocols to measure the affinity of odorants and pheromones to soluble olfactory proteins, such as odorant-binding proteins, chemosensory proteins and Niemann-Pick class C2 proteins.

The invention relates generally to biosensors for detecting an analyte in a sample.

In an aspect of the invention, there is provided a biosensor as recited in Claim <NUM>, the biosensor comprising:.

Preferably, the biosensor further comprises:.

In another aspect of the invention, there is provided use of the biosensor, wherein the biosensor is used to identify olfactory ligands.

In another aspect of the invention, there is provided use of the biosensor, wherein the biosensor is used in high-throughput screening.

In another aspect of the invention, there is provided use of the biosensor, wherein the biosensor is used to detect field populations of aphids.

In another aspect of the invention, there is provided an in vitro method for detecting an analyte in a sample as recited in Claim <NUM>, the method comprising:.

Preferably, the biosensor is used to identify olfactory ligands.

Preferably, the biosensor is used in high-throughput screening.

Preferably, the biosensor is used to detect field populations of aphids.

The invention is further described by reference to the following non-limiting figures.

The present invention will now be further described. In the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of microbiology, tissue culture, molecular biology, chemistry, biochemistry, recombinant DNA technology and biosensor fabrication, which are generally known in the art. Such techniques are explained fully in the literature.

Olfactory proteins are responsible for the detection of odorants and may therefore be suitable for the detection of sex pheromones associated with insects, for example aphids.

Olfactory proteins may include odorant receptors (ORs). Non-limiting examples of odorant receptors include odorant receptor <NUM>/olfactory receptor co-receptor (OR1/ORCO), odorant receptor <NUM> (OR2), odorant receptor <NUM> (OR4), odorant receptor <NUM> (OR5), odorant receptor <NUM> (OR10), odorant receptor <NUM> (OR17), odorant receptor <NUM> (OR20), odorant receptor 22c (OR22c), odorant receptor <NUM> (OR23), odorant receptor <NUM> (OR25), odorant receptor <NUM> (OR31), odorant receptor <NUM> (OR37), odorant receptor <NUM> (OR38),odorant receptor <NUM> (OR39), odorant receptor <NUM> (OR42) and odorant receptor <NUM> (OR43).

Olfactory proteins may include olfactory binding proteins (OBPs). Non-limiting examples of olfactory binding proteins include olfactory binding protein <NUM> (OBP1), olfactory binding protein <NUM> (OBP2), olfactory binding protein <NUM> (OBP3), olfactory binding protein <NUM> (OBP4), olfactory binding protein <NUM> (OBP5), olfactory binding protein <NUM> (OBP6), olfactory binding protein <NUM> (OBP7), olfactory binding protein <NUM> (OBP8), olfactory binding protein <NUM> (OBP9), olfactory binding protein <NUM> (OBP10) and olfactory binding protein <NUM> (OBP11).

The biosensor comprises Acyrthosiphon pisum OBP6 (having an amino acid sequence as defined in SEQ ID NO: <NUM>), or a variant thereof.

The term "variant" or "functional variant" as used herein with reference to any of the sequences described herein refers to a variant polypeptide sequence or part of the polypeptide sequence which retains the biological function of the full non-variant sequence. A functional variant also comprises a variant of the polypeptide of interest, which has sequence alterations that do not affect function, for example in non-conserved residues. Also encompassed is a variant that is substantially identical, i.e. has only some sequence variations, for example in non-conserved residues, compared to the wild type sequences as shown herein and is biologically active. Alterations in a polypeptide sequence that does not affect the functional properties of the polypeptide are well known in the art. For example, the amino acid alanine, a hydrophobic amino acid, may be substituted by another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine. Similarly, changes which result in substitution of one negatively charged residue for another, such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine, can also be expected to produce a functionally equivalent product. Each of the proposed modifications is well within the routine skill in the art, as is determination of retention of biological activity of the encoded products.

As used in any aspect described herein, a "variant" or a "functional variant" has at least <NUM>% (for example <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, or at least <NUM>%) overall sequence identity to the non-variant amino acid sequence.

Preferably, a "variant" or a "functional variant" as described herein has at least <NUM>% (for example at least <NUM>%, <NUM>%, <NUM>% or <NUM>%) overall sequence identity to the non-variant amino acid sequence.

It is particularly preferred that a "variant" or a "functional variant" as described herein has at least <NUM>%, <NUM>% or <NUM>% overall sequence identity to the non-variant amino acid sequence.

Two polypeptides are said to be "identical" if the sequence of amino acid residues, respectively, in the two sequences is the same when aligned for maximum correspondence as described below. The terms "identical" or percent "identity", in the context of two or more polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues that are the same, when compared and aligned for maximum correspondence over a comparison window, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. When percentage of sequence identity is used in reference to proteins or peptides, it is recognised that residue positions that are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. Non-limiting examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST <NUM> algorithms.

In an embodiment, the biosensor may further comprise a signal generator. For example, the signal generator may be a transducer.

The signal generator may be configured to output a signal when the analyte has bound to the olfactory protein.

The signal generator may be configured to output a different signal when the analyte is not bound to the olfactory protein.

The signal generator may be configured to output no signal when the analyte is not bound to the olfactory protein.

A skilled person would also understand that a situation where the analyte has not bound to the olfactory protein leads to an output of a signal, and where the analyte has bound to the olfactory protein leads to an output of no signal, would be embraced by the present disclosure.

A magnitude of the signal generated may be correlated or proportional to the concentration of analyte in the sample. For example, a higher concentration of analyte may be associated with a more intense signal in terms of magnitude; conversely, a lower concentration of analyte may be associated with a less intense signal in terms of magnitude.

The signal may be based on an electrical signal. For example, the signal may be a potential difference, a current, a resistance, a capacitance or an impedance associated with the electrical signal.

The signal may be based on an optical signal. For example, the signal may be based on various properties of electromagnetic radiation, such as wavelength, intensity, absorption, scattering or fluorescence. For example, the electromagnetic radiation may be ultraviolet light, visible light or infrared light.

The biosensor may operate based on electrochemical means. Binding of an analyte may cause consumption or generation of electrons. Alternatively, binding of an analyte does not cause direct electron flow but causes changes on an electrode surface, for example, changes in charge, hydration or pH.

Non-limiting examples of an electrochemical-based biosensor include impedimetric, amperometric, potentiometric, conductometric and voltametric biosensors. An electrochemical-based biosensor may include biosensor field-effect transistors (BioFETs), such as ion-sensitive field-effect transistors (ISFETs).

The biosensor may operate based on optical means. For example, the biosensor may operate in a label-free mode, where an optical signal is generated directly by the interaction of the analyte with a transduction element. Alternatively, the biosensor may operate with a label and an optical signal is generated by a colorimetric, fluorescent or luminescent method.

Non-limiting examples of an optical-based biosensor include surface plasmon resonance (SPR), evanescent wave fluorescence, optical waveguide interferometric, ellipsometric, reflectometric interference spectroscopy (RIfS) and surface-enhanced Raman scattering (SERS) biosensors.

The biosensor may operate based on piezoelectric means. For example, the biosensor may include a piezoelectric crystal (e.g. quartz) which vibrates under the influence of an electric field. The resonant frequency may change as an analyte adsorbs or desorbs from the surface of the piezoelectric crystal.

In an embodiment, the biosensor may further comprise a flow path for moving a sample, wherein the sample may contain an analyte.

In an embodiment, the biosensor may further comprise a substrate.

For example, the substrate may be a glass substrate or a polymer substrate. The substrate may exhibit excellent mechanical strength, thermal stability, transparency, surface smoothness, ease of handling, and water resistance.

In an embodiment, the biosensor may further comprise a protein-containing layer immobilised to the substrate, wherein the protein-containing layer contains the olfactory protein. The protein-containing layer may be in contact with the flow path.

Means for immobilising proteins onto a substrate are well known in the art. Non-limiting methods of immobilising proteins include adsorption, cross-linking, covalent bonding, electrochemical polymerisation and photopolymerisation.

In an embodiment, the biosensor may further comprise a working electrode.

In an embodiment, the biosensor may further comprise a counter electrode.

The working electrode and/or the counter electrode may be partially exposed at the flow path and utilised for applying a voltage to a sample.

The working electrode and/or the counter electrode may comprise an electrically conductive material. An electrically conductive material is any material that allows the flow of charge (electrical current) in one or more directions. Non-limiting examples of electrically conductive materials may include metals and metal alloys, such as gold, platinum, copper, silver, aluminium, palladium, steel, brass and bronze; carbon-based materials, such as graphite, single-wall carbon nanotubes (SWCNTs), multi-wall carbon nanotubes (MWCNTs), graphene, graphene oxide; metal oxides, such as indium tin oxide (ITO), indium zinc oxide (IZO), tin oxide, zinc oxide; and organic conducting polymers, such as polythiophene, polyaniline and polypyrrole.

In an embodiment, the biosensor may further comprise a reference electrode.

The reference electrode may provide a reference signal, for example, a stable potential, relative to the sample. For example, a reference electrode may be an Ag/AgCl electrode.

Biosensors as described herein may have high binding affinity to the sex pheromones of insects and therefore may be highly sensitive to the sex pheromones of insects.

For example, biosensors as described herein may be highly sensitive to the sex pheromones of aphids, and in particular, Acyrthosiphon pisum.

Biosensors as described herein may be highly sensitive to the presence of nepetalactols or nepetalactones.

For example, biosensors as described herein may be highly sensitive to the presence of (<NUM>R,4aS,<NUM>S,7aR)-nepetalactol ((<NUM>R,4aS,<NUM>S,7aR)-<NUM>,<NUM>-dimethyl-<NUM>,4a,<NUM>,<NUM>,<NUM>,7a-hexahydrocyclopenta[c]pyran-<NUM>-ol), (4aS,<NUM>S,7aR)-nepetalactone ((4aS,<NUM>S,7aR)-<NUM>,<NUM>-dimethyl-<NUM>,<NUM>,<NUM>,7a-tetrahydrocyclopenta[c]pyran-<NUM>(4aH)-one), (<NUM>S,4aR,<NUM>R,7aS)-nepetalactol ((<NUM>S,4aR,<NUM>R,7aS)-<NUM>,<NUM>-dimethyl-<NUM>,4a,<NUM>,<NUM>,<NUM>,7a-hexahydrocyclopenta[c]pyran-<NUM>-ol) and/or (4aR,<NUM>R,7aS)-nepetalactone ((4aR,<NUM>R,7aS)-<NUM>,<NUM>-dimethyl-<NUM>,<NUM>,<NUM>,7a-tetrahydrocyclopenta[c]pyran-<NUM>(4aH)-one).

In particular, the biosensors as described herein may be highly sensitive to the presence of natural nepetalactols or nepetalactones, such as (<NUM>R,4aS,<NUM>S,7aR)-nepetalactol and (4aS,<NUM>S,7aR)-nepetalactone.

As used herein, the term "highly sensitive" may refer to a biosensor comprising an olfactory protein as described herein, or a variant thereof, having a binding energy of -<NUM> kcal mol-<NUM>, preferably -<NUM> kcal mol-<NUM>, more preferably -<NUM> kcal mol-<NUM>, even more preferably -<NUM> kcal mol-<NUM>, yet even more preferably -<NUM> kcal mol-<NUM>, most preferably -<NUM> kcal mol-<NUM>, to a nepetalactol or nepetalactone as described herein.

Biosensors as described herein may generally be used for the detection of analytes.

In an embodiment, the biosensors described herein may be used to identify new olfactory ligands.

For example, the new olfactory ligands may be associated with aphids, and in particular, Acyrthosiphon pisum.

For example, the biosensor may be used as a lab screening tool to identify new olfactory ligands. For example, the biosensor may be used to identify aphid attractants other than sex pheromone components.

In an embodiment, the biosensors described herein may be used in high-throughput screening.

High-throughput screening (HTS) typically uses automated assays to search through large numbers of compounds for a desired activity. High throughput methods enable researchers to assay thousands of different chemicals against each target molecule very quickly using robotic handling systems and automated analysis of results.

As used herein, "high throughput screening" or "HTS" refers to the rapid in vitro screening of large numbers of compounds (libraries); generally tens to hundreds of thousands of compounds, using robotic screening assays. Ultra high-throughput screening (uHTS) generally refers to the high-throughput screening accelerated to greater than <NUM>,<NUM> tests per day.

To achieve high-throughput screening, it is advantageous to house samples on a multicontainer carrier or platform. A multicontainer carrier facilitates measuring reactions of a plurality of candidate compounds simultaneously. Multi-well microplates may be used as the carrier. Such multi-well microplates, and methods for their use in numerous assays, are both known in the art and commercially available.

In an embodiment, the biosensors described herein may be used to detect field populations of aphids.

For example, the biosensor may be deployed as a field tool.

In an embodiment, an in vitro method of detecting an analyte in a sample is provided, comprising: (a) providing a biosensor as described herein; (b) contacting the biosensor with the sample; and (c) comparing a magnitude of the signal generated by the biosensor when the sample is present with a reference magnitude of the signal generated by the biosensor when the sample is absent.

The following non-limiting examples are provided for further illustration.

All previously published protein structures were accessed from the Protein Data Bank (PDB).

Sequence alignments were performed using Cluster Omega and the PRALINE server. Conservation mapping was performed using ConSurf. Transmembrane domain predications of the receptor proteins were performed using a consensus approach and four different serves - HMMTOP, TMpred, PHOBIUS and TMHMM. Alignments and conservation maps were analysed in GeneDoc, and phylogenetic trees were generated in FigTree v1.

For odorant-binding proteins, protein structures were initially predicted using the iTASSER database, which takes a hierarchical approach by identifying structural templates from the Protein Data Bank. All predicted protein structures were minimised using the Yasara minimisation server.

For odorant-receptors, Homology based modelling was performed using the olfactory receptor co-receptor, ORCO, from Apocrypta bakeri as a template. The structure of AbakORCO was obtained from the Protein Data Bank (PDB ID 6C70; resolution <NUM>Å). Pairwise sequence alignment was performed using PRALINE and the predicted transmembrane domains were manually aligned and annotated. The available ORCO structure shares a generally low sequence identity with the A. pisum odorant receptors, however, the general seven transmembrane structure should be conserved. The pairwise alignment served as a template for homology modelling using MODELLER <NUM> with loop refinement. The secondary structure of long extracellular loops <NUM> and <NUM> (EL2 and EL3) were individually predicted using the iTASSER server. Approximately <NUM> models were generated for each protein, and these were subsequently assessed using discrete optimised protein energy (DOPE) from MODELLER, in addition to Ramachandran and Z-score analysis, performed in PROCHECK and ProSA respectively.

All protein structures were visualised in PyMol <NUM>.

Ligands were prepared in Chem3D <NUM> and AutoDock <NUM> (Python Molecule Viewer), then screened against computer generated models using AutoDock4. <NUM> and the Racoon virtual screening tool. A Lamarckian Genetic Algorithm was selected for the simulation.

OBPs and ORs were all screened against a wide range of ligands. The binding energy of the complex and subsequent Ki were calculated.

Molecular dynamics simulations were performed using GROMACS <NUM>. For odorant-binding proteins, the OLPS force field was used, and for odorant-receptors, a modified Gromas 53a6 force field was utilised.

For odorant-binding proteins, models were solvated and neutralised with ions (for negative Cl-, for positive Na+) before energy minimisation and equilibration (temperature and pressure) calculations were performed. For each protein, a <NUM> ns simulation was performed. The stability of the protein was then assessed by observing temperature and pressure stabilisation of the simulated models.

Molecular dynamics simulations were performed using the GROMACS <NUM> package. For OBPs, and OLPS force field was used, whereas for odorant-receptors, the Gromos53a6 forcefield was utilised, with modifications made to the Gromos53a6 forcefield parameters to include lipids and lipid topology. Lipid bilayer topology and structure were obtained from http://wcm. ca/tieleman/downloads. Receptor models were embedded into a lipid bilayer of <NUM> dipalmitoylphosphatidylcholine (DPPC) molecules. All models were solvated with explicit solvent (SPC) water and neutralised with either Cl- or Na+ ions. Steepest-descent methods were used to minimise the energy of each system, at a reference temperature of <NUM>. All bonds were constrained with LINCS and long-range electrostatics were handled using PME.

Structures of Compounds <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM>-<NUM>, Compound A ((<NUM>R,4aS,<NUM>S,7aR)-nepetalactol), Compound B ((4aS,<NUM>S,7aR)-nepetalactone), Compound C ((<NUM>S,4aR,<NUM>R,7aS)-nepetalactol), Compound D ((4aR,<NUM>R,7aS)-nepetalactone) and Compound E ((<NUM>R,<NUM>S,<NUM>S)-dolichodial) are provided below.

Structures of Compounds <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM>-<NUM> and Compounds A-E:
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>.

Referring to Table <NUM>, the compounds of Examples <NUM> to <NUM> (reference) have a strong binding affinity to wild type Acyrthosiphon pisum OBP6. The compounds of Examples <NUM> to <NUM> also have high binding affinity compared with the compounds of Comparative Examples <NUM> to <NUM>.

<FIG> shows that the lone pair on the oxygen atom within the six-membered ring of natural nepetalactone is held close to another oxygen lone pair on Tyr176. Without wishing to be bound by theory, it is postulated that a change from an oxygen atom to a carbon, nitrogen or sulfur atom (e.g. Examples <NUM>-<NUM> and <NUM>-<NUM> (reference)) helps to increase binding affinity as there is less electrostatic repulsion (in the case of carbon and nitrogen, the lone pair is removed; in the case of sulfur, the lone pair is more diffuse with a less electronegative sulfur atom). It is also postulated that in the case of sulfur, the larger size of the sulfur atom leads to puckering of the ring system away from the oxygen lone pair on Tyr176, further enhancing binding affinity. Other structural changes (e.g. in Examples <NUM>, <NUM> and <NUM>) also help to increase binding affinity.

Referring to Table <NUM>, OBP6 may have strong binding affinity to the aphid sex pheromone components, for both the biologically active enantiomers and the inactive enantiomers.

Media for use in molecular biology experiments were prepared as in Table <NUM>.

Media was sterilised by autoclaving at <NUM>, then cooled before adding an appropriate antibiotic to a working concentration (Table <NUM>). Agar was kept at <NUM> until poured into plates and allowed to set.

All genes were previously cloned into a vector plasmid, pET45b or pET15b, containing an ampicillin resistance cassette, or pNIC28-Bsa4, containing a kanamycin resistance cassette. Each plasmid contained a hexa-histidine (His<NUM>) tag encoded at the N-terminus of the protein, to allow for nickel affinity purification.

To transform cells, <NUM>µL of plasmid was added to BL21(DE3) competent E. coli cells and cooled to <NUM>. The mixture was allowed to sit for <NUM> minutes, after which it was heat-shocked at <NUM> for <NUM> sec and cooled to <NUM> for a further <NUM> minutes. LB liquid media (<NUM>µL) was added to the sample, followed by incubation (<NUM> rpm; <NUM>) for <NUM> minutes. The sample was centrifuged (<NUM> rpm) for <NUM> minutes and the pellet resuspended in LB media (<NUM>µL). The resuspension was spread onto an LB-agar plate containing the appropriate antibiotic and incubated (<NUM>) for <NUM> hours. A clonal cell template for PCR was made from the overnight culture by diluting a scraping from a small, single colony in H<NUM>O (<NUM>µL).

A mixture for PCR was made by combining the cell template (<NUM>µL), appropriate forward and reverse primers (<NUM>µL of each), H<NUM>O (<NUM>µL) and a Taq polymerase mix (<NUM>µL) containing dNTP, Taq polymerase and PCR buffer (<NUM> Tris-HCl, <NUM> KCl, <NUM> MgCl<NUM>, pH <NUM>). The mixture was heated at <NUM> for <NUM> minutes, followed by <NUM> cycles of heating at <NUM> for <NUM> seconds, to <NUM> for <NUM> seconds, then <NUM>. Finally, the mixture was taken to <NUM> for <NUM> minutes and stored at <NUM>. Gel electrophoresis with a <NUM>% agarose gel was used to check the PCR product and determine whether the transformation was successful.

A scraping from clonal culture or glycerol stock sample (<NUM>µL) was added to 2xYT liquid media (<NUM>) and incubated overnight (<NUM>; <NUM> rpm) to generate a starter culture of E. coli BL21(DE3).

Starter culture of recombinant BL21(DE3) E. coli cells (<NUM>µL) was added to 2xYT liquid media containing the appropriate antibiotic (<NUM>) and incubated (<NUM>; <NUM> rpm) until the OD<NUM> values reached <NUM>-<NUM>. Isopropyl β-D-<NUM>-thiogalactopyranoside (IPTG; <NUM>µL; <NUM>) was added to induce expression, and the mixture incubated for a further <NUM>-<NUM> hours (<NUM>; <NUM> rpm).

A sample of the culture (<NUM>) was removed and centrifuged (12000rpm; RT) for <NUM>, and the cell pellet resuspended in SDS-PAGE loading buffer (600µL). Samples were then denatured at <NUM> for <NUM> minutes, before loading onto an SDS-PAGE gel.

2xYT liquid media (<NUM>) was inoculated with starter culture (<NUM>) and incubated (<NUM>, <NUM> rpm) until an OD<NUM> of <NUM>-<NUM> was reached. IPTG (<NUM>µL; <NUM>) was added to induce expression and the mixture incubated for a further <NUM>-<NUM> hours (<NUM>, <NUM> rpm). Cells were harvested by centrifuging for <NUM> minutes (<NUM> rpm).

The cell pellet was resuspended in ice-cold TBS buffer (<NUM>; <NUM> Tris, <NUM> NaCl, pH <NUM>) and incubated on ice for <NUM> minutes. The mixture was sonicated for <NUM> minutes on ice, then centrifuged for <NUM> minutes (<NUM> rpm, <NUM>). The pellet was resuspended in TBS+<NUM>% Triton X-<NUM> (<NUM>). The sonication and centrifugation steps were repeated, and the pellet resuspended in <NUM> urea (<NUM>) and <NUM> <NUM>,<NUM>-dithiothreitol in <NUM> Tris (<NUM>; pH <NUM>). The mixture was incubated for <NUM> hour at room temperature, then rapidly diluted with <NUM> TBS+<NUM>:<NUM> GSH:GSSG. The diluted mix was incubated overnight at RT.

The overnight mixture was centrifuged for <NUM> minutes (<NUM> rpm) and the supernatant filtered through a <NUM> filter.

OBPs were purified using Histrap® columns, preconditioned with <NUM> imidazole buffer. The filtrate from the refolding step (Chapter <NUM>; <NUM>. <NUM>) was passed through the column, leaving the histidine-tagged protein bound to the nickel. An imidazole buffer (<NUM>) was used to displace the protein and fractions were collected and analysed using SDS-PAGE.

The resolving gel (<NUM> width; <NUM>) was prepared as in Table <NUM>. Approximately <NUM> of <NUM>-isopronaol was added to remove bubbles and the gel left to set. After <NUM> minutes, the isopropanol was removed, and the stacking gel added. A comb was used to form wells before leaving the gel to set for a further <NUM> minutes.

Samples for the gel (<NUM>µL) were prepared by combining protein samples (<NUM>µL) with sample buffer (<NUM>µL; prepared as in Table <NUM>). Samples were run alongside an appropriate marker (<NUM>-<NUM> kDa). The gel was run at <NUM> V for <NUM>-<NUM> minutes and GelCode™ Blue Stain (Thermo-Fisher Scientific) was added for approximately <NUM> hour to resolve the protein bands.

To remove the hexa-histidine tag from purified proteins, an appropriate cleavage enzyme was used (Table <NUM>). The selection of the enzyme depended on the cleavage site encoded in the original vector plasmid. The protein was buffer-exchanged in CaCl<NUM> in TBS buffer (<NUM> CaCl<NUM>, <NUM> Tris; <NUM> NaCl), and enzyme added. Cleavage was monitored by observing a change in mass using mass spectrometry. After completion, excess tagged protein was removed by passing the mixture through a Histrap® column and collecting the eluent.

Plasmids were extracted using a GeneJET Plasmid Miniprep Kit (Thermo-Fisher Scientific) and sequenced by Queen's Medical Centre laboratories, University of Nottingham or Eurofins (UK).

All proteins were buffer exchanged and desalted using a PD-<NUM> desalting column and concentrated using a Vivaspin <NUM> (<NUM> kDa MWCO).

Site-directed mutagenesis (SDM) of OBP6-His<NUM> was performed to insert a thrombin tag. A Q5 Site-Directed Mutagenesis kit (New England Biolabs) was used to perform the mutagenesis. Primers (Table <NUM>) were obtained from Sigma-Aldrich.

Samples for PCR were prepared by combining plasmid containing the gene of interest (<NUM>µL), forward and reverse primers (<NUM>µL of each), H<NUM>O (<NUM>µL) and Q5 hot start master mix (<NUM>µL). The mixture was heated at <NUM> for <NUM> seconds, followed by <NUM> cycles of heating at <NUM> for <NUM> seconds, to <NUM> for <NUM> seconds, then <NUM> for <NUM> minutes. Finally, the mixture was taken to <NUM> for <NUM> minutes and stored at <NUM>.

The PCR product (<NUM>µL) was mixed with KLD reaction buffer (<NUM>µL), KLD enzyme mix (<NUM>µL) and H<NUM>O (<NUM>µL) and left at RT for <NUM> minutes. Finally, the mixture (<NUM>µL) was used to transform NEB <NUM>-alpha Competent E. coli cells as described under "Transformation of E. coli BL21(DE3) Competent Cells with A. pisum OBP Plasmids". Results were validated by sequences as described under "Plasmid Extraction, Purification and Sequencing".

To purify proteins, gel filtration via fast-protein liquid chromatography (FPLC) was performed using an Akta FPLC. The FPLC was fitted with a Superdex size-exclusion column, and samples were exchanged into a TBS buffer (Tris <NUM>, NaCl <NUM>; Table <NUM>). Protein samples were collected using an autosampler and subsequently concentrated as described under "Protein Buffer Exchange and Concentration".

To confirm efficient cleavage of the His-tag and determination of the structure, denatured mass-spectrometry of the recombinant proteins was performed using a Waters QTof2 spectrometer. Samples were prepared using a ZipTip® before injected into the mass spectrometer.

All mass spectrometry was undertaken using electrospray ionisation and conducted on the QTof2 or QTof3 under denaturing conditions (<NUM>% CH<NUM>CN, <NUM>% formic acid) and the settings detailed in Table <NUM>, unless otherwise stated.

Mass spectra were analysed using MassLynx <NUM> software.

Samples for mass-spectrometry were prepared by ZipTip®. The ZipTip® was conditioned by washing with elution buffer (<NUM>% CH<NUM>CN, <NUM>% formic acid; 5x10 µL), followed by equilibrating with wash buffer (<NUM>% MeOH, <NUM>% trifluoroacetic acid; 7x10 µL). Trifluoroacetic acid (TFA) was added to the sample to a concentration of <NUM>%, which was adsorbed onto the column (<NUM> aspirations). The sample was then washed with wash buffer (20x10 µL) and eluted into elution buffer (<NUM>µL, <NUM> aspirations).

All fluorescent measurements were undertaken using a Perkin-Elmer LS50B fluorescence spectrophotometer, using a <NUM> quartz cuvette, and the settings described below in Table <NUM>, unless otherwise stated. Spectra were recorded using FL WinLab software.

Saturation of OBPs with fluorescent probe and <NUM>-NPN (Sigma-Aldrich) was initially measured by titrating a <NUM> protein sample (<NUM> in <NUM> Tris-HCl) with aliquots of <NUM> ligand (Sigma-Aldrich) in methanol to final concentrations of <NUM>-<NUM>. The fluorescence intensity at <NUM> was recorded.

The competitive binding of ligands was measured by observing the intrinsic fluorescence of tryptophan. Titrations were performed with aliquots of <NUM> ligand in methanol to final concentrations of <NUM>-<NUM>, either after the addition of fluorescent probe to a final concentration of <NUM> or in the absence of fluorescent probe.

To generate KD values, relative fluorescence intensity was plotted against the concentration of ligand as a binding curve. KD values were generated in GraphPad Prism <NUM> using a non-linear regression and the inbuilt equation:
<MAT>.

Each calculated KD value had an associated error from the non-linear regression. To account for these errors in statistical analysis, values were weighted in direct proportion to their error - the higher the error, the lower the weighting of the values. The 'weight' factor was calculated using the following equation:
<MAT>.

Statistical analysis of fluorescence data was performed using R <NUM>. A one-way weighted ANOVA was performed between ligands for each protein, and a two-way weighted ANOVA was performed to investigate the interactions between proteins and ligands. A Tukey Test was used for post-hoc analysis (Table <NUM>).

The interaction between (4aS,<NUM>S,7aR)-nepetalactone and (4aR,<NUM>R,7aS)-nepetalactone did not give a significant difference, with a p-value of <NUM> in the <NUM>-NPN assay and a p-value of <NUM> in the fluorescent probe free assay. A weighted t-test was subsequently performed, giving a significant difference with a p-value of <NUM> x <NUM>-<NUM>.

Samples were run using an AVANCE Bruker DRX-<NUM> Nuclear Magnetic Resonance spectrometer with a <NUM> BBO BB-<NUM> probe and set at <NUM> for <NUM>H spectra and <NUM> for <NUM>C spectra. Analysis of Bruker data was performed using Topspin <NUM>. <NUM>, and analysis of Varien data performed with CCPNMR V2 and NMRPipe. Standard NMR tubes were used with a sample volume of <NUM>µL unless otherwise stated. Deuterated chloroform (CDCl<NUM>), methanol (CD<NUM>OD) and dimethyl sulfoxide (d-DMSO) were stored over <NUM>Å molecular sieves and used as both sample solvents and internal standards. For assignment of small molecules, additional 2D-NMR spectroscopy experiments were performed.

Protein samples for NMR were desalted and buffer exchanged into <NUM>:<NUM><NUM>O:D<NUM>O unless stated otherwise (as in "Protein Buffer Exchange and Concentration"). Ligand samples were not soluble in D<NUM>O and samples were prepared in d-DMSO. This resulted in a final NMR solvent including H<NUM>O, D<NUM>O and d-DMSO.

An initial test assay containing Bovine Serum Albumin (BSA) protein with tryptophan and sucrose was prepared for STD NMR. Samples were run for <NUM> scans, and the saturation transfer difference (STD) spectra generated.

STD absolute values were calculated by observing the change in proportions between the off-resonance spectrum and the final STD spectrum using the following equation:
<MAT>
in which the term (I<NUM> - ISTD) represents the ratio of peak intensity in the STD spectrum and I<NUM> the ratio of intensity in the off resonance spectrum. A second value representing the proportionate change was calculated using the following equation:
<MAT>.

Finally, epitope mapping was performed by calculating the relative peak integration in the STD spectrum vs the off-resonance spectrum of a peak as a percentage.

Nepetalactone and derivatives may be synthesized using (inverse electron demand) Diels-Alder reactions as described in <NPL>) and <NPL>). Some further modifications are illustrated below.

Ammonia gas was bubbled gently through a solution of nepetalactone (<NUM>, <NUM> mmol) in DCM (<NUM>) for <NUM> hour. The reaction flask was then sealed and stirred for a further <NUM> hours. The crude reaction mixture was concentrated under vacuum and purified via distillation (Kugelrohr, <NUM> mbar, <NUM>) to yield the product. δH (<NUM>, CDCl<NUM>) <NUM> (<NUM>, s), <NUM> - <NUM> (<NUM>, m), <NUM> (<NUM>, q, J = <NUM>), <NUM> - <NUM> (<NUM>, m), <NUM> - <NUM> (<NUM>, m), <NUM> - <NUM> (<NUM>, m), <NUM> (<NUM>, s), <NUM> - <NUM> (<NUM>, m), <NUM> (<NUM>, d, J = <NUM>).

In order, indium triflate (<NUM>, <NUM> mmol), toluene (<NUM>), nepetalactone (<NUM>, <NUM> mmol), sulphur (<NUM>, <NUM> mmol) and phenyl silane (<NUM>, <NUM> mmol), under N<NUM>, was added to a screw topped flask, sealed and heated to <NUM> for <NUM> hours. The reaction mixture was cooled and the pressure released carefully. The reaction mixture was filtered through celite before being purified on silica gel (<NUM>% EtOAc in hexanes) to give the product (<NUM>, <NUM>% yield) as yellow/brown oil. δH (<NUM>, CDCl<NUM>) <NUM> (<NUM>, s), <NUM> - <NUM> (<NUM>, m), <NUM> (<NUM>, hept, J = <NUM>), <NUM> (<NUM>, t, J = <NUM>), <NUM> - <NUM> (<NUM>, m), <NUM> (<NUM>, s), <NUM> - <NUM> (<NUM>, m), <NUM> - <NUM> (<NUM>, m), <NUM> (<NUM>, d, J = <NUM>).

To a solution of nepetalactone (<NUM>, <NUM> mmol) in acetonitrile (<NUM>) was added phosphorus pentasulphide (<NUM>, <NUM> mmol) followed by hexamethyldisiloxane (<NUM>, <NUM> mmol) and heated to <NUM> for <NUM> hours. The solvent was removed under vacuum and the crude product purified on silica gel (<NUM>% EtOAc in pet ether) to give the product (<NUM>, <NUM>% yield) as a yellow oil. δH (<NUM>, CDCl<NUM>) <NUM> (<NUM>, s), <NUM> (<NUM>, t, J = <NUM>), <NUM> - <NUM> (<NUM>, m), <NUM> (<NUM>, hept, J = <NUM>), <NUM> - <NUM> (<NUM>, m), <NUM> - <NUM> (<NUM>, m), <NUM> (<NUM>, s), <NUM> - <NUM> (<NUM>, m), <NUM> (<NUM>, d, J = <NUM>).

To a solution of nepetathionolactone (Compound <NUM>) (<NUM>, <NUM> mmol) in isopropyl alcohol (<NUM>) was added sodium borohydride (<NUM>, <NUM> mmol) and the mixture stirred for <NUM> hours. The reaction was quenched with <NUM> of <NUM> HCl and saturated brine was added. The aqueous layer was extracted with diethyl ether. The combined organic layer was dried (MgSO<NUM>) and concentrated under vacuum. The crude product was purified on silica gel (<NUM>% Et<NUM>O in pet ether) to give the product (<NUM>, <NUM>% yield) as a pale yellow oil. δH (<NUM>, CDCl<NUM>) <NUM> (<NUM>, s), <NUM> (<NUM>, d, J = <NUM>), <NUM> (<NUM>, q, J = <NUM>), <NUM> - <NUM> (<NUM>, m), <NUM> - <NUM> (<NUM>, m), <NUM> (<NUM>, s), <NUM> - <NUM> (<NUM>, m), <NUM> (<NUM>, d, J = <NUM>).

A sensing surface (a film bulk acoustic wave resonator - FBAR - fabricated by Sorex Sensors) was functionalised via the APTES-glutaraldehyde method. The sensing surface to be functionalised was exposed to a stream of ozone for <NUM> mins, before being allowed to stand in air for <NUM> mins and washed with distilled water. To the freshly oxidised sensing surface, a solution of APTES (<NUM>% in ethanol) was added and ensured the whole sensing surface was covered and allowed to stand for <NUM> mins. The excess reagent was removed with clean ethanol, before being dried under a stream of nitrogen and cured in an oven (<NUM>) for <NUM> mins. After cooling to RT, a <NUM>% solution of glutaraldehyde was added to the surface for a further <NUM> mins. A solution of the desired OBP (ApisOBP6, <NUM>) was added to the surface for <NUM> hour. The functionalised sensing surface was washed with distilled water and stored until required.

While the foregoing disclosure provides a general description of the subject matter encompassed within the scope of the present invention, including methods, as well as the best mode thereof, of making and using this invention, the following examples are provided to further enable those skilled in the art to practice this invention and to provide a complete written description thereof. However, those skilled in the art will appreciate that the specifics of these examples should not be read as limiting on the invention, the scope of which should be apprehended from the claims and equivalents thereof appended to this disclosure. Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.

"and/or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example "A and/or B" is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.

Claim 1:
A biosensor for detecting an analyte in a sample, the biosensor comprising:
a protein having an amino acid sequence as defined in SEQ ID NO: <NUM>, or a variant thereof, wherein the variant has at least <NUM>% overall sequence identity to SEQ ID NO: <NUM>; and
a signal generator, wherein the signal generator is configured to output a signal when the analyte is bound to the protein.