Methods for identifying compounds that interact with olfactory receptors

Abstract

The invention provides methods for associating volatile compounds or mixtures of volatile compounds with combinatorial response patterns of Olfactory Receptors (OlfRs), a biosensor array comprising a plurality of biosensors, the use of said biosensor and of the method of the invention to screen for diseases, a molecule identified by a method of the invention as well as the use se of said molecule to modify the behavior of an insect.

Description

[0001] Unitec Ref: 1189-B1557 PCT FINAL PAT8401PC00

[0002] Methods for identifying compounds that interact with olfactory receptors

[0003] FIELD OF THE INVENTION

[0004] The invention provides methods for associating volatile compounds or mixtures of volatile compounds with combinatorial response patterns of Olfactory Receptors (OlfRs), a biosensor array comprising a plurality of biosensors, the use of said biosensor and of the method of the invention to screen for diseases, a molecule identified by a method of the invention as well as the use of said molecule for influencing the behavior, mating and / or feeding habits of an insect.

[0005] BACKGROUND OF THE INVENTION

[0006] Insecticides are crucial for maintaining agricultural productivity by effectively managing pests that threaten crop yields. Without effective pest control, agriculture faces significant losses; it is estimated that insect pests can destroy approximately 20-40% of global crop production annually. This makes insecticides vital in ensuring a consistent food supply, and thus food security.

[0007] However, the broad application of these chemicals is linked with significant environmental and health risks. Insecticides can contaminate soil and waterways, causing longterm ecological disturbances. Their persistence means that residues can linger in the environment, entering food chains and presenting ongoing risks to both wildlife and human health. For consumers, this results in exposure to potentially harmful chemical residues on and in food products, which can lead to various health issues, ranging from acute poisoning to chronic diseases. Moreover, insecticides indiscriminately negatively affect many organisms, not just the targeted pests. This action contributes to a decline in insect biodiversity, potentially leading to a massive extinction event that could destabilize entire ecosystems. Insects are integral to nutrient cycling, pollination, and as food sources for other species, making their decline a grave ecological threat.

[0008] Despite these significant challenges, alternatives to insecticides often lack the efficacy, scalability, or cost-effectiveness of chemical treatments, leaving the agricultural sector heavily Unitec Ref: 1189-B1557 PCT FINAL PAT8401PC00 reliant on insecticides. Adding further pressure, regulatory changes and pesticide bans in response to environmental and health concerns are increasingly common, compelling farmers to seek alternative pest control methods that may not yet provide the same level of effectiveness or may be more costly to implement than insecticides.

[0009] This situation underscores the critical need for innovative solutions in pest control. A promising emerging approach to manage pest populations in agriculture is the use of volatile compounds to manipulate pest insect behaviors. This method leverages the olfactory systems of insects, which insects rely on for various behaviors crucial to their survival, such as finding food, mates, and suitable locations for laying eggs. Insects detect chemical signals through their olfactory subsystems (including antennae and palps), which contain receptors that can identify a wide range of volatile compounds. By understanding and manipulating these olfactory cues, scientists can devise strategies that repel pests from crops, attract them to traps, prevent proper egg laying or disrupt their reproduction. For instance, certain volatile compounds can be used to create repellent barriers around seeds, growing or mature plants, or harvest, deterring pests from feeding or laying eggs. Alternatively, attractants can lure pests into traps where they can be captured or exposed to insecticides in a confined and controlled environment, reducing the need to apply chemicals over a large area. Such an approach represents a number of advantages. First, it is targeted to specific species, minimizing collateral damage to non-target species, including beneficial insects like pollinators and natural predators of pests. These approaches allow the reduction of chemical use, are safer for the environment and human health, and can be used in combination with other pest management strategies.

[0010] While the application of volatile compounds in pest control shows significant promise, the principal challenge lies in identifying compounds that effectively influence insect behavior while remaining cost-effective and environmentally safe. Many natural odors that trigger insect behaviors, such as certain sexual pheromones or volatile compounds involved in oviposition and predator avoidance, are often hard to identify and costly to produce. Understanding which volatile compounds are detected by pest insects and how these modulate behavior is crucial to finding cost-effective alternatives.

[0011] Insects generally express an extensive array of olfactory receptor (OlfR) genes — ranging from several dozen to hundreds — in specialized organs in larvae, and in their antennae and palps in adults. Typically, each olfactory sensory neuron (OSN) expresses one or very few OlfR. genes that are from one of two genetically unrelated families: the odorant receptor (OR) Unitec Ref: 1189-B1557 PCT FINAL PAT8401PC00 and the ionotropic receptor (IR) families. OSNs project their dendrite in hair-like structures called sensilla that cover the surface of the sensory organs. These sensilla are filled with mucus and a number of proteins that allow the transport and degradation of volatile compounds. Specific behaviors are initiated by the activation of either a single OlfR or a specific combination of OlfRs. To identify appropriate ligands or combinations of ligands that can induce specific behaviors, it is crucial to determine which receptors are activated by which volatile compounds. For instance, by pinpointing which receptors are stimulated by naturally occurring complex volatile compound blends inducing specific behaviors, researchers can create inexpensive synthetic chemicals that either mimic or block these signals. Mimicking these signals can lure pests into traps or trigger avoidance, while blocking them can make crops less detectable to pests. These approaches offer efficient and less harmful alternatives to traditional pesticides. A method for more comprehensive and rapid screening of OlfR responses against a variety of volatile compounds is key to devising more targeted, sustainable, and economically feasible pest control strategies using volatile compounds.

[0012] The identification of volatile compound signatures is not only relevant for developing pest control approaches and can have other uses for the general detection of volatile molecules. For example, it could be of interest to detect volatile compounds that are indicative of the presence of dangerous or illicit substances. Moreover, it is well established that certain pathological conditions, including cancers and metabolic disorders, produce characteristic volatile molecules that can be detected through smell, as demonstrated, for example, by trained dogs. The success of these animals serves as a proof of concept that the detection of volatile compounds can be used to screen for pathologies; however, screening with trained animals is complicated and expensive, and therefore difficult to implement broadly in a medical context.

[0013] Current approaches to identifying OlfR-ligand pairs encompass both in vitro and in vivo methodologies. In vitro approaches primarily rely on heterologous expression systems such as HEK293 cells, Sf9 cells, and Xenopus oocytes. These approaches often face difficulties in expressing a significant subset of these receptors. For those expressed, their agonist responses often poorly recapitulate their in-vivo characteristics, leading to an incomplete or inaccurate reproduction of OlfR native responses. Other techniques, such as electro-antennograms, record from entire antennas and allow the recording of the global neuronal activity within an antenna, but do not allow to attribute the response to one or another of the many OlfRs expressed in the antenna. These approaches are therefore of not much use when one needs to associate a specific volatile compound to specific OlfRs. Unitec Ref: 1189-B1557 PCT FINAL PAT8401PC00

[0014] The main approach to identify OlfR-ligand pairs in vivo relies on the "empty neuron" technique in Drosophila melanogaster. This method involves removing endogenous receptors in drosophila OSNs to introduce exogenous insect OlfRs. While this allows for studying OlfRs in a more biologically relevant context, it introduces significant throughput challenges. To each OlfR of the repertoire must correspond a single transgenic fly line expressing the OlfR in the empty neuron. Then, for each OlfR to be tested, the response requires an independent single neuron electrophysiological recording performed with a technique called single sensillum recording (SSR). This technique entails targeting individual sensilla with a recording electrode and measuring the responses of the neurons it harbors. Thus, experimenters must always make sure to target a sensillum harboring the dendrite of the empty neuron expressing the exogenous OlfR. Consequently, testing a single volatile compound against an OlfR repertoire requires performing a series of SSRs equal to the size of the repertoire to be tested, severely limiting the speed and scalability of this approach. This represents a major bottleneck in high- throughput screening for OlfR-volatile compound pairs.

[0015] Recently, a different approach using the empty neuron technique has been proposed, involving the co-expression of an exogenous olfactory receptor (OlfR) with GCaMP to assess in vivo OlfR activation via live trans-cuticular calcium imaging. This method allows for limited parallel recording of OlfR activity, with reports of up to two live Drosophila being recorded simultaneously, and the suggestion that up to four flies could be recorded in a single experiment. This limitation stems from the requirement to keep all antennas within the microscope's field of view and on the same focal plane while ensuring they remain functional, which means it is necessary to keep the entire flies alive with their antennas exposed. Considering that an insect's OlfR repertoire can range from dozens to hundreds of receptors, this method is not suitable for studying the sensitivity of large OlfR repertoires that recapitulate the olfactory abilities of an insect species, as it can only assess up to a maximum of four OlfRs at a time.

[0016] This highlights a pressing need for methods with increased throughput in which a large number of samples can be tested simultaneously.

[0017] SUMMARY OF THE INVENTION

[0018] The present invention provides a biosensor array comprising a plurality of biosensors, wherein each biosensor comprises a severed transgenic Drosophila head or olfactory sensory organ (antenna and / or maxillary palp) of said transgenic Drosophila positioned so that at least Unitec Ref: 1189-B1557 PCT FINAL PAT8401PC00 one antenna and / or maxillary palp is in contact with air, and the head or olfactory sensory organ is in contact with a nutritious and hydrating matrix, and wherein the cells of the antenna and / or the maxillary palp lack endogenous Olfactory Receptors (OlfRs) and co-express at least one exogeneous insect OlfR and a genetically encoded neuronal activity reporter.

[0019] Further provided is a method for associating a volatile molecule or mixture of volatile compounds with a combinatorial response pattern of OlfRs, the method comprising the steps of: a) providing a biosensor array of the invention, b) contacting the biosensor array with a volatile compound or mixture of volatile compounds, c) detecting the neuronal activity in parallel in each individual biosensor of the biosensor array upon exposure to the volatile compound / molecule or mixture of volatile compounds, whereby a change of the neuronal activity in an individual biosensor compared to a baseline indicates a response of the exogenous OlfR expressed in said biosensor to the volatile compound / molecule or mixture of volatile compounds, and d) determining the combinatorial response pattern of OlfRs associated with said volatile compound / molecule or mixture of volatile compounds.

[0020] Further provided are one or more volatile compounds or mixtures of volatile compounds identified according to a method of the invention.

[0021] The invention further provides a method for diagnosing and / or prognosing a disease in a subject comprising the steps of: a) providing a biosensor array according to the invention, b) contacting the biosensor array with a biological sample derived from the subject, c) detecting the neuronal activity in parallel in each individual biosensor of the biosensor array upon exposure to the volatile molecule or mixture of volatile compounds of the biological sample of step b), whereby a change of the neuronal activity in an individual biosensor compared to a baseline indicates a response of the exogenous OlfR expressed in said biosensor to a volatile molecule or mixture of volatile compounds, d) determining the combinatorial response pattern of OlfRs associated with said volatile molecule or mixture of volatile compounds of the biological sample, Unitec Ref: 1189-B1557 PCT FINAL PAT8401PC00 e) comparing the combinatorial response pattern of OlfRs to the volatile molecule or mixture of volatile compounds of the biological sample to a reference sample, wherein a differential combinatorial response pattern of OlfRs to the volatile molecule or mixture of volatile compounds of the biological sample relative to the corresponding combinatorial response pattern of OlfRs to the volatile molecule or mixture of volatile compounds of the reference sample is indicative of a diagnosis and / or a prognosis of said disease in the subject.

[0022] The invention further provides a method of treatment of a disease in a subject, comprising the steps of: a) identifying OlfRs with a differential combinatorial response pattern indicative of a diagnosis and / or a prognosis of a disease in the subject according to the invention, b) treating the subject based upon the differential combinatorial response pattern of the identified OlfRs indicative of a diagnosis and / or a prognosis of a disease in the subject.

[0023] The invention further provides a kit for performing the methods according to the invention, said kit comprising a) the biosensor array of the invention, b) means and / or reagents for determining the combinatorial response pattern of OlfR(s), and / or c) instructions for use.

[0024] The invention further provides the use of the kit of the invention in a method for the detection of the presence, absence and / or amount of volatile molecule or mixture of volatile compounds in a test sample or biological sample compared to a reference sample according to the invention.

[0025] The invention further provides the use of the kit of the invention in a method for screening, diagnosing or prognosing a disease in a subject of the invention.

[0026] The invention further provides the use of the kit of the invention in a method of treatment of a disease in a subject of the invention. Unitec Ref: 1189-B1557 PCT FINAL PAT8401PC00

[0027] DESCRIPTION OF THE FIGURES

[0028] Figure 1: Description of the biosensor array, a) Schematic of the empty neuron approach associated with the addition of a neuronal activity reporter, in which defined olfactory sensory neurons are depleted from their endogenous chemoreceptor gene, and in which an exogenous olfactory receptor is transgenically added, together with a neuronal activity reporter, b) Schematic representation of a biosensor array made of an organotypic culture of Drosophila heads on a nutrient-rich matrix, ready for parallel OlfR activation recording, c) Schematic representation of a biosensor array made of an organotypic culture of Drosophila antennae on a nutrient-rich matrix, ready for parallel OlfR activation recording.

[0029] Figure !: Specificity, concentration-dependent sensitivity, robustness, and stability of the biosensor array, a) Calcium imaging responses of DmOR22a and DmOR98a upon exposure to a series of volatile compounds. Volatile compound stimulations consist of three one-second pulses, each separated by one-minute intervals, b) Calcium imaging response of DmOR59b to varying concentrations of methyl acetate, c) Serial activation of DmOR59b by methyl acetate at a dilution of 10e3. d) Volatile compound activation test on long-term organotypic cultures (Oh, 24h, and 144h) of Drosophila heads. The y-axis is set to 0 at the time of stimulation. For panels b), c), and d), the traces shown are normalized using a moving AF / F. 2,3 BDO (2,3 butanediol), EtHex (ethyl hexanoate), Eug (eugenol), 1-Oct (l-octe-3-ol), BuAc (butyl acetate).

[0030] Figure 3: Combinatorial response patterns of OlfRs from different insect species a) Neuronal responses tested for 225 volatile compound-DmOR pairs, b) Responses of two Helicoverpa armigera olfactory receptors (HarmOR42 and HarmOR40) to a set of volatile compounds.

[0031] Figure 4: Activation and inhibition of IRs and OlfRs to volatiles using the biosensor array a) Response of DmIR84a / DmIR8a to its ligand phenylacetaldehyde, b) Inhibition of DmOR59b basal activity by linalool as well as inhibition of DmOR59b agonist-induced (ethyl acetate) activity by linalool.

[0032] Figure 5: Detection and discrimination of volatile compounds associated with urine of healthy and sick individuals with four selected Delia platura olfactory receptors. a) Responses of four Delia platura ORs to Urine of LPS injected (sick) and saline injected (heatlhy) mice. Each sample corresponds to a pool of urine collected from 5 mice. Unitec Ref: 1189-B1557 PCT FINAL PAT8401PC00 b) t-SNE visualization of olfactory receptor responses distinguishing urine samples from healthy and sick mice. Each point represents the response pattern of four olfactory receptors to one tested sample. Points are colored according to the health status of the mice.

[0033] DESCRIPTION OF THE INVENTION

[0034] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The publications and applications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.

[0035] In the case of conflict, the present specification, including definitions, will control. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in art to which the subject matter herein belongs. As used herein, the following definitions are supplied in order to facilitate the understanding of the present invention.

[0036] The term "comprise / comprising" is generally used in the sense of include / including, that is to say permitting the presence of one or more features or components. The terms "comprise(s)" and "comprising" also encompass the more restricted ones "consist(s)", "consisting" as well as "consist / consisting essentially of', respectively.

[0037] As used in the specification and claims, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise.

[0038] As used herein, the terms "co-express" or "co-expression" refer to the production of more than one protein product, at the same time, from the genetic information contained within one or more nucleic acid sequence(s).

[0039] As used herein, the terms "protein", "peptide", "polypeptide" and "polypeptide fragment" are used interchangeably herein to refer to polymers of amino acid residues of any Unitec Ref: 1189-B1557 PCT FINAL PAT8401PC00 length. The polymer can be linear or branched, it may comprise modified amino acids or amino acid analogs, and it may be interrupted by chemical moi eties other than amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, PEGylation or any other manipulation or modification, such as conjugation with a labeling or bioactive component.

[0040] As used herein, the terms "repertoire" " refers to a set of genes encoding a plurality of different OlfRs. The full OR repertoire of Drosophila melanogaster was first described in Vosshall, L. B., Amrein, H., Morozov, P. S., Rzhetsky, A., & Axel, R. (2000). A spatial map of olfactory receptor expression in the Drosophila antenna. Cell, 96(5), 725-736. DOI: 10. 1016 / s0092-8674(00)80582-6 and Clyne, P. J., Warr, C. G„ Freeman, M. R„ Lessing, D„ Kim, J., & Carlson, J. R. (1999). A novel family of divergent seven-transmembrane proteins: candidate odorant receptors in Drosophila. Neuron, 22(2), 327-338. DOI: 10.1016 / s0896- 6273(00)81093-4. The first description of the IR repertoire of Drosophila melanogaster was published in Benton, R., Vannice, K. S., Gomez-Diaz, C., & Vosshall, L. B. (2009). Variant ionotropic glutamate receptors as chemosensory receptors in Drosophila. Cell, 136(1), 149-162. DOI: 10.1016 / j .cell.2008. 12.001. In some aspects, the repertoire represents all of the Olfactory Receptors of an insect species. In some aspects, the repertoire represents the Olfactory Receptors that detect a volatile compound or a mixture of volatile compounds. In some aspects, the repertoire represents the OlfRs that detect a desired, attractant, or adverse, repellent taste, scent, smell, and / or odor. In some aspects, the repertoire represents the OlfRs of a class, family, or type. In some aspects, the repertoire represents the set of OlfRs expressed in a given sensory organ.

[0041] The term “repellent”, as used herein, refers to a repellent effect against insects which reduces the infestation of insects and / or controls and / or combats any population of insects. It is preferred within the present invention that a reduction of infestation of at least 20%, 25%, 30%, 35%, 40, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% is achieved when compared to the case where no repellent is present. Within the present invention, reducing the infestation may be achieved through the repelling activity of the composition or compound of the invention.

[0042] The term “attractant”, as used herein, refers to an attractant effect on insects. It is preferred within the present invention that a n increase of attraction of at least 20%, 25%, 30%, Unitec Ref: 1189-B1557 PCT FINAL PAT8401PC00

[0043] 35%, 40, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% is achieved when compared to the case where no attractant is present. Within the present invention, the increase may be achieved through the attracting activity of the composition or compound of the invention.

[0044] The term “about,” particularly in reference to a given quantity, number, temperature or percentage, is meant to encompass deviations of plus or minus ten percent (± 10). For example, about 5% encompasses any value between 4.5% to 5.5%, such as 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, or 5.5.

[0045] A “plurality of biosensors” refers to at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100 or more biosensors as defined herein.

[0046] The present invention provides a biosensor array comprising a plurality of biosensors, wherein each biosensor comprises a severed transgenic Drosophila head or olfactory sensory organ (antenna and / or maxillary palp) of said transgenic Drosophila positioned so that at least one antenna and / or maxillary palp is in contact with air, and the head or olfactory sensory organ is in contact with a nutritious and hydrating matrix, and wherein the cells of the antenna and / or the maxillary palp lack endogenous Olfactory Receptors (OlfRs) and co-express at least one exogenous insect OlfR and a genetically encoded neuronal activity reporter.

[0047] The present invention provides a biosensor array comprising a plurality of biosensors, wherein each biosensor comprises a severed transgenic Drosophila head or olfactory sensory organ (antenna and / or maxillary palp) of said transgenic Dr osophila in contact with a nutritious and hydrating matrix, and wherein the sensory neurons of the antenna and / or the maxillary palp lack endogenous Olfactory Receptors (OlfRs) and co-express at least one exogeneous insect OlfR and a genetically encoded neuronal activity reporter.

[0048] In one aspect of the invention, co-expression of an exogenous OlfR and an activity reporter is driven in a single population of antennal or maxillary palp neurons, defined by the endogenous OlfR gene they typically express but lacking this receptor due to a targeted knockout. In another aspect, co-expression of an exogenous OlfR and activity reporter is driven across multiple populations of antennal or maxillary palp sensory neurons each lacking expression of their respective endogenous OlfR. This broader targeting increases the global signal emitted when the exogenous OlfR is activated. Unitec Ref: 1189-B1557 PCT FINAL PAT8401PC00

[0049] In one aspect, the at least one antenna and / or maxillary palp is in contact with air and the severed head or olfactory sensory organ is in contact with a nutritious and hydrating matrix.

[0050] In one aspect of the invention the sensory organs ( i.e. the antenna the palp or the whole head) of the transgenic drosophila co-expressing specific exogenous OlfRs and the neuronal activity reporter are separated from the rest of the body and arranged in contact with a gelatinous matrix, a liquid layer or a porous material providing a liquid media to sustain organotypic culture. This allows maintaining the olfactory sensory organ alive and functional while exposing it to compounds and recording its potential neuronal response to these volatile compounds.

[0051] Drosophila antennae are approximately 100 microns wide and 200 microns long, which makes it challenging to position them in contact with a liquid medium without submerging them, a situation that would hinder subsequent exposure to volatile compounds. Thus, placing them on a gelatinous medium solution is a convenient setup.

[0052] It is crucial for the open circulatory system containing hemolymph to be in contact with the culture medium to preserve the functionality of the olfactory sensory organs after resection. An effective way to achieve this is by placing the cut site directly in contact with the medium. Alternatively, for antennae, they can also be placed laterally on the medium. When using an entire Drosophila head, it can be positioned with the antennae facing upward, ensuring that the open circulatory system and hemolymph are in contact with the medium while leaving the antennae exposed for recording.

[0053] Preferably, the matrix comprises a nutritious and hydrating medium selected from the group comprising balanced salt solutions, amino acid(s), vitamin(s), buffers, carbohydrate(s), serum or serum replacements, hydrolysates and growth factor(s), or a combination thereof. Nonlimiting examples of commercial nutritious and hydrating medium comprise Schneider's insect medium suitable for insect cell culture (sold e.g. by Thermo Fischer Scientific or Sigma Aldrich, see Schneider, Imogene (1964) Exp. Zool. 156: 1 :91), D-20 medium or D-22 medium (Echalier G, Ohanessian A. In Vitro. 1970 Nov-Dec;6(3): 162-72.)

[0054] In one aspect, the matrix is gelatinous, a thin liquid layer, or a porous material.

[0055] In one aspect of the invention, the nutritious and hydrating matrix is provided in the form of a gelatinous matrix, a thin layer of liquid, or a liquid in a porous matrix and comprises balanced salt solutions, amino acids, vitamins, buffers, serum or serum replacements, hydrolysates and growth factors, or a combination thereof. Unitec Ref: 1189-B1557 PCT FINAL PAT8401PC00

[0056] In the array, each of these biosensors may express a different exogenous OlfR. Alternatively, biosensors expressing the same exogenous OlfR can be replicated multiple times within the array. Replication helps to reduce variability and serves as a technical control.

[0057] The use of isolated olfactory sensory organs (antenna and / or maxillary palp) or heads as biosensors is an essential aspect of the invention as it allows for packing numerous (at least 5) biosensors in a single biosensor array. Increasing the number of biosensors allows for increasing the number of OlfRs that are tested in parallel in a single volatile compound exposure or mixture of volatile compounds exposure.

[0058] In one aspect, the plurality of biosensors of the invention comprises at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100 or more drosophila heads or olfactory sensory organs.

[0059] For example, where an array is composed of 100 biosensors, an organized matrix can be set (10x10 for example), or the biosensors may be arranged in a less organized matrix, providing that the location of each biosensor identity of the sensing unit within the pattern is known. For a 10x10 matrix of heads, the size of the biosensor array would be about lOmmxlOmm. For a 10x10 array of antennas, the dimensions would be around 5mmx5mm. Any arrangement allowing to pack many sensory organs in a single biosensor is preferred.

[0060] The biosensor array remains functional for hours at least up to 2 hours, at least up to 5 hours, at least up to 10 hours of continuous testing and can be stored for several days, or at least up to 1 week at a cool temperature (approx. 4-6°C).

[0061] A trained experimenter can set up a biosensor array of 10x10 biosensors in less than one hour. The process includes putting the flies to sleep, placing them under a microscope and sectioning the sensory organs or heads with a scalpel, and placing the olfactory sensory organs or heads on the air-liquid interface culture system. This process can be automatized with a robotic arm to further decrease the time necessary for setting up the biosensor array.

[0062] In one aspect, the biosensor array of the invention comprises an entire OlfR repertoire of at least one insect.

[0063] In one aspect of the invention an entire OlfR repertoire of one insect species endogenously expressed in a specific olfactory organ or antenna palp of the adult, or the dorsal organ of the Unitec Ref: 1189-B1557 PCT FINAL PAT8401PC00 larvae of this species, can be present in a single biosensor array. In another aspect of the invention a subset of OlfRs can be present in the biosensor array. In yet another aspect, OlfRs from different species may be present in a single biosensor array.

[0064] In one aspect of the invention, the section point of the severed head or olfactory sensory organ is specifically put in contact with the nutritious and hydrating medium. This allows for the hemolymph to be in direct contact with the culture medium.

[0065] The medium used to keep the olfactory organs alive and functional corresponds to any medium that could be usually used for insect tissue culture, such as e.g. Schneider, D-20, or Grace medium. These media are designed to mimic insect hemolymph. Other media mimicking hemolymph are also expected to work. These media contain a mixture of amino acids, vitamins, inorganic salts, sugars (like glucose), and other nutrients required to support cell growth and viability. These media provide the essential nutrients for insect cells to function and stay alive, mimicking their physiological environment. These media contain balanced salt solutions that help maintain osmotic pressure, pH balance, and proper ion concentrations necessary for cell metabolism. They are formulated to maintain stable pH under typical insect cell culture conditions (pH around 6.2 to 6.4). The media can be supplemented with fetal bovine serum (FBS) or other growth factors to promote robust cell survival and function.

[0066] The media can be delivered in the form of a gelatinous matrix, for example agarose, a thin layer of liquid, or through porous cell culture support membranes which are thin, permeable membranes used in cell culture systems to provide a surface for cells to grow while allowing the exchange of nutrients, gases, and other molecules between different compartments of the culture system. These membranes are typically used in transwell or insert systems.

[0067] In one aspect of the invention, the biosensor arrays are maintained at a controlled temperature suitable to the function and survival of the cultured olfactory sensory organs, usually between 19 and 25 degrees Celsius.

[0068] In one aspect, the biosensor array or plurality of biosensors is functional for at least 2 days, preferably at least 3 days, more preferably at least 5 days and even more preferably at least 7 days or more.

[0069] In one aspect, the biosensor array or plurality of biosensors can be stored at about 0°C to about 10°C for at least 2 days, preferably at least 5 days, more preferably at least 7 days and even more preferably at least 10 days or more. Unitec Ref: 1189-B1557 PCT FINAL PAT8401PC00

[0070] In one aspect, the biosensor array or plurality of biosensors can be stored at about -80°C to about -10°C for at least 2 days, preferably at least 5 days, more preferably at least 7 days and even more preferably at least 10 days or more.

[0071] As used herein, the term "insect" includes any stage of development of an insect, including a one-celled germ line cell, a fertilized egg, an early embryo, a larva, including any of a first through a later instar larva, a pupa, or an adult insect. This invention utilizes transgenic host insects, in particular Drosophila species, for the expression of exogenous olfactory receptors (OlfRs) selected from various insect species. The sensitivity of these exogenous OlfRs to volatile compounds is tested.

[0072] In one aspect of the invention, the at least one exogeneous OlfR belongs to at least one insect. In one aspect of the invention, the at least one exogeneous OlfR belongs to at least one insect, preferably selected from the group comprising an insect that i) is harmful to agricultural crops, ii) presents a safety risk for humans or animals and / or iii) causes a nuisance for humans.

[0073] In one aspect of the invention, the exogeneous OlfR belongs to at least one insect selected from the group comprising an insect that i) is harmful to agricultural crops, ii) presents a safety risk for humans or animals and / or iii) causes a nuisance for humans.

[0074] The exogenous OlfRs tested can be selected from a variety of insect species. In one aspect, the insect providing the exogenous OlfRs is selected from the group comprising Archaeognatha, Blattodea, Coleoptera, Dermaptera, Diptera, Embioptera, Ephemeroptera, Grylloblattodea, Hemiptera, Hymenoptera, Lepidoptera, Mantodea, Mantophasmatodea, Mecoptera, Megaloptera, Neuroptera, Odonata, Orthoptera, Phasmatodea, Phthiraptera, Plecoptera, Psocodea, Psocoptera, Raphidioptera, Siphonaptera, Strepsiptera, Thysanoptera, Trichoptera, Zoraptera, Isoptera, Notoptera, Protura, Diplura, and Zygentoma, or a combination thereof.

[0075] In one aspect, the insect providing the exogenous OlfRs pertains to the species Schistocerca gregaria, Spodoptera frugiperda, Nilaparvata lugens, Helicoverpa armigera, Plutella xylostella, Bemisia tabaci, Myzus persicae, Chilo suppressalis, Leptinotarsa decemlineata, Drosophila suzukii, Diaphorina citri, Sitophilus oryzae, Ceratitis capitata, Cydia pomonella, Phthorimaea operculella, Frankliniella occidentalis, Diabrotica virgifera virgifera, Nezara viridula, Maconellicoccus hirsutus, Lymantria dispar, Spodoptera exigua, Tuta absoluta, Locusta Unitec Ref: 1189-B1557 PCT FINAL PAT8401PC00 migratoria, Cosmopolites sordidus, Hypothenemus hampei, Rhynchophorus ferrugineus, Rhagoletis pomonella, Bactrocera cucurbitae, Bactrocera dorsalis, Ostrinia nubilalis, Diatraea saccharalis, Anarsia lineatella, Rhyzopertha dominica, Sitobion avenae, Diuraphis noxia, Pieris rapae, Pectinophora gossypiella, Rhopalosiphum maidis, Cephus cinctus, Delia platura, Schizaphis graminum, Stenodiplosis sorghicola, Mythimna unipuncta, Aphis glycines, Psila rosae, Delia platura, Oulema melanopus, Leucoptera coffeella, Lissorhoptrus oryzophilus, Lipaphis erysimi, Duponchelia fovealis, Dysdercus peruvianus, Cacopsylla pyricola, Phyllocnistis citrella, Pseudococcus maritimus, Phenacoccus manihoti, Aspidiotus destructor, Stemochetus mangiferae, Cerataphis brasiliensis, Cylas formicarius, Pianococcus citri, Agrotis ipsilon, Orseolia oryzae, Hypera postica, Spodopteralittoralis, Thrips tabaci, Aphis craccivora, Thaumetopoea pityocampa, Helopeltis theivora, Scirpophaga incertulas, Sitophilus zeamais, Liriomyza sativae, Idioscopus clypealis, Apion frumentarium, Nipaecoccus nipae, Tetranychus urticae, Ectomyelois ceratoniae, Apion stellulatum, Blissus leucopterus, Chrysodeixis includens, Helicoverpa zea, Sogatella furcifera, Zygogramma exclamationis, Euproctis chrysorrhoea, Sesamia calamistis, Nephotettix virescens, Tetranychus cinnabarinus, Bactrocera invadens, Conotrachelus nenuphar, Ascia monuste, Heliothis virescens, Cnaphalocrocis medinalis, Aulacaspis yasumatsui, Sitotroga cerealella, Anthonomus grandis, Macrosiphum euphorbiae, Apion mediotinctum, Diachasma alloeum, Chaetocnema tibialis, or Scirtothrips dorsalis.

[0076] In one aspect, the host insect is from the order Diptera. Preferably the insect is a drosophila species selected from the group comprising Melanogaster subgroup (e.g. D. melanogaster, D. simulans, D. teissieri, D. yakuba, D. erecta, and D. orena), Ananassae subgroup, Drosophila majtoi, Elegans subgroup, Eugracilis subgroup, Ficusphila subgroup, Flavohirta subgroup, and Montium subgroup, or a combination thereof. More preferably, the drosophila is a D. melanogaster.

[0077] In certain aspects of the invention, the method involves utilizing the "empty neuron" strategy, as described in [Chahda JS, Soni N, Sun JS, Ebrahim SAM, Weiss BL, Carlson JR. The molecular and cellular basis of olfactory response to tsetse fly attractants. PLoS Genet. 2019 Mar 15;15(3):el008005. doi: 10.1371 / joumal.pgen.l008005. PMID: 30875383; PMCID: PMC6420007] and [Kurtovic, A., Widmer, A. & Dickson, B. A single class of olfactory neurons mediates behavioral responses to a Drosophila sex pheromone. Nature 446, 542-546 (2007). https : / / doi. org / 10, 1038 / nature05671 ■ Unitec Ref: 1189-B1557 PCT FINAL PAT8401PC00

[0078] The "empty neuron" system can be employed to express at least one exogenous OlfR and a genetically encoded neuronal activity reporter such as e.g. a genetically encoded calcium indicator (GECI) or a genetically encoded voltage indicator (GEVI). This system is based on mutant antennal and / or maxillary palp neurons that lack endogenous odorant receptors, rendering them unresponsive to odors (Dobritsa et al., 2003; Mariette et al., 2023), unless an exogenous OlfR is forcibly expressed in these neurons. In one aspect of the present invention, OlfRs are typically expressed, or co-expressed, in these empty neurons alongside a GECI for functional studies.

[0079] Practically, in the host insect, the coding sequence (CDS) of an olfactory receptor (OlfR) is knocked out and a GAL4 CDS is placed under the control of the knocked out OlfR promoter, effectively creating olfactory sensory empty neurons. These neurons allow for the coexpression of an exogenous OlfR and a genetically encoded neuronal activity reporter (under the control of an upstream activating sequence (UAS) that binds to GAL4), trans genically inserted in the host Drosophila genome.

[0080] Methods for generating transgenic insects are well-established in the art. In one aspect, transgenic flies are generated by using CRISPR / Cas9 to insert the GAL4 CDS into OlfR CDSs, thereby knocking out the OlfR and thus generating a population of empty neurons amenable for transgene expression using UAS promoters.

[0081] In another aspect, genes encoding for proteins such as an exogenous OlfR and a neuronal activity reporter are placed under the control of an expression system, such as UAS, and cloned into a suitable vector, for example, a plasmid containing an attB sequence. This construct is then introduced into insect eggs (e.g., via microinjection), and the transgene(s) are integrated into the genome at an attP site through phiC31 -mediated recombination, as described by Jack R. Bateman et al. (2006), Genetics 173(2): 769-777.

[0082] Other systems, such as TetON / TetOFF or qUAS, may be used alternatively to achieve the coexpression exogenous OlfRs and activity markers.

[0083] As used herein, the terms "Olfactory Receptor" or "OlfR" are used interchangeably herein to refer to olfactory receptors. OlfR encompasses two families of transmembrane proteins: odorant receptors (ORs) and ionotropic receptors (IRs). The terms “odorant receptors” and “ORs” are used interchangeably herein to refer to odorant receptors. ORs are ligand-gated ion channels composed of a divergent odorant receptor and a conserved co-receptor (Oreo). These receptors are expressed in olfactory sensory neurons of both larvae and adult insects. Specific Unitec Ref: 1189-B1557 PCT FINAL PAT8401PC00 odorant receptors are usually abbreviated using the genus and species abbreviations (such as Dm for Drosophila melanogaster) followed by the term OR and the ID number of the receptor that can be complemented with a letter, for example, DmOR59b).

[0084] The terms “ionotropic receptors” and “IRs” are used interchangeably herein to refer to ionotropic receptors. IRs are ligand-gated ion channels comprising a divergent odorant receptor and one or more co-receptors. Like ORs, they are expressed in olfactory sensory neurons of larvae and adult insects.

[0085] In some aspects, the olfactory receptors used in the invention are exogeneous OlfRs, i.e. OlfRs not endogenously expressed in the host insect species in which they are transgenically driven.

[0086] It is understood that any olfactory receptor of any insect is encompassed in the present invention.

[0087] The odorant receptor (OlfR) coding sequence (CDS) repertoire for a given insect species can be retrieved from genomic repositories or browsers, such as the NCBI Genome Database (https: / / www.ncbi.nlm.nih.gov / datasets / genome) or ENSEMBL Metazoa (https: / / metazoa.ensembl.org).

[0088] These genomes may be pre-annotated, or in certain cases, manual annotation may be required by conducting BLAST searches against known OlfR sequences. In the absence of publicly available genome assemblies, whole-genome sequencing (WGS) or RNA sequencing (RNA- Seq) of olfactory tissues (such as antennae) can be performed to capture OlfR CDSs, enabling de novo assembly and identification of OlfR CDSs.

[0089] Usually, the molecule of interest is a volatile compound, and may originate from a solid or liquid substrate. It can be natural, synthetic, known, unknown, provided in pure form or as part of a blend. Mixtures of volatile compounds are also envisioned in the present invention.

[0090] In one aspect, the tested compounds may come from human bodily secretions, for example, to identify insect receptors that respond to them. Alternatively, the biosensor array can detect specific volatile compounds present in human secretions.

[0091] Volatile compounds responsible for triggering insect behaviors, both innate and learned, are also included. These include pheromones, kairomones, apneumones, allomones, or other infochemicals. The volatile compounds could, for instance, be associated with germinating seeds, oviposition substrates, food, toxic substances, or insect predators. Unitec Ref: 1189-B1557 PCT FINAL PAT8401PC00

[0092] Volatile compounds can be applied manually to the biosensor array, for instance, using a pipette filled with the volatile compounds, or with an olfactometer. Using an olfactometer offers several advantages, such as standardizing stimulation pulses. Additionally, olfactometers often have multiple channels that can be pre-loaded, allowing the automatic testing of a series of volatile compounds or their blends. These volatile compounds can be pure, part of a blend, or originate from solid or liquid samples.

[0093] Volatile compounds can be collected by different methods. In static headspace sampling (HS), volatile compounds naturally migrate into the gas phase above the sample. Alternatively, in dynamic headspace sampling (DHS), an inert gas is passed over the sample to extract and concentrate the volatile compounds. Solid phase microextraction (SPME) involves exposing a fiber coated with adsorbent material to the liquid or headspace, capturing volatile compounds that are later thermally desorbed and applied to the biosensor array. Thermal desorption (TD) can be used for natural samples by heating them and thermally desorbing the volatile compounds into the gas phase. For solid samples, direct sublimation is another option, where heating causes sublimation of volatile compounds, which are then captured or transferred directly to the biosensor array.

[0094] The preferred technique for sampling volatile compounds is static headspace sampling. In this method, samples containing volatile compounds are placed in containers, such as vials, and transported to the biosensor array via tubing. The volatile compounds should be applied to the biosensor array at a temperature between 10°C and 30°C, which can be adjusted based on the volatility of the compounds.

[0095] Volatile compounds or their blends or mixtures can be loaded into the olfactometer either in pure or diluted form. Dilution is usually done with water, mineral oil, paraffin oil, ethanol, hexane, acetone, pentane, isopropanol, dipropylene glycol, or DMSO, depending on the polarity of the compound. Solid compounds may need to be dissolved in solvents to promote volatilization. Volatile compound pulses are delivered using commercial devices, like the Aurora220 olfactometer, or a custom-made system. Dilutions can be prepared in advance, and the intervals between pulses typically range from 1 second to several minutes, with 30 seconds to 1 minute being common due to the dynamics of calcium imaging. Pulse durations can vary from fractions of a second to up to 20 seconds. Unitec Ref: 1189-B1557 PCT FINAL PAT8401PC00

[0096] It is understood that any method for detecting an increase or decrease of calcium flux is encompassed in the present invention. It is understood that the detection method will depend on the reporter used as described below.

[0097] In one aspect, sensory cells of the antenna and / or the maxillary palp co-express at least one exogeneous OlfR and a genetically encoded neuronal activity reporter selected from the nonlimiting group comprising a genetically encoded calcium indicator (GECI), and a genetically encoded voltage indicator (GEVI), or a combination thereof. When an appropriate interaction occurs at the OlfR (e.g. binding of molecule to the OlfR), the reporter is activated. In one aspect of the invention, the olfactory sensory neurons triggered by the binding of a volatile compound to the exogenous OlfR is detected through changes of fluorescence of a GECI directly through the cuticula of the cultured olfactory sensory organs. This technique is called transcuticular calcium imaging, see Mariette et al., 2023.

[0098] In some aspects, the sensory cells are engineered to co-express a single activity reporter and an exogenous OlfR. In someaspects, different sensory cell populations, each corresponding to a different endogenous OlfR that has been removed, are targeted together to enhance signal detection. In some embodiments, a sensory cell is engineered to express two or more reporter products, for example by using a single vector construct encoding two or more reporters.

[0099] As used herein, the term " neuronal activity reporter" refers to any moiety capable of reflecting neuronal activity, and whose signal may be detected indirectly or directly.

[0100] In one aspect, the genetically encoded neuronal activity reporter is selected from the group comprising a genetically encoded calcium indicator (GECI), and a genetically encoded voltage indicator (GEVI), or a combination thereof.

[0101] In one aspect of the invention, the neuronal activity reporter is a GECI, in particular a GCaMP. GCaMPs are a family of GECIs in which the calcium-binding domains are attached to one circularly permuted GFP molecule. Their structure is based on the calmodulin (CaM) calcium binding domain as the sensing element, which interacts with the calcium-CaM-binding motif Ml 3 from the Myosin Light Chain Kinase protein (MCLK) and the circularly permuted GFP. The calcium ion-binding protein according to the invention is not particularly limited as long as the effects of the present invention are shown.

[0102] In one aspect, the invention provides a GECI selected from the group comprising GCaMP, R- GECOls, GEM-GECO, and Chameleon, a variant or a combination thereof. Unitec Ref: 1189-B1557 PCT FINAL PAT8401PC00

[0103] Usually, GCaMPs consist of three key domains: a calcium-binding protein calmodulin (CaM), a peptide sequence from myosin light-chain kinase that interacts with CaM (Ml 3), and a reporter such as for example a green fluorescent protein (GFP).

[0104] Examples thereof can include fluorescent proteins which are bound to calcium ion typified by Geco such as G-Geco, B-Geco, R-Geco, GEX-Geco or GEM-Geco; Case 12, CEPIA, Aequorin, Cameleon, Pericam or GcaMP or the like (R-CaMP). Preferably, the GECI is selected from the group comprising GCaMP or a variant thereof, R-GECOls, GEM-GECO, and Cameleon, or a combination thereof.

[0105] Methods for measuring the fluxes of calcium inside cells using the calcium ion-binding proteins disclosed herein are not limited. Specifically, examples thereof can include a method for measuring fluorescence values emitted by a calcium ion-binding protein depending on the binding state of the calcium ion-binding protein and calcium ion after being brought into contact. An advantage of the biosensor array is that commercially available tools can be used to record and measure intracellular calcium transients. Typically, fluorescence can be visualized with a standard fluorescence microscope. Microscopes with large fields of view are preferable. The position of the sensory organs can be manually or automatically assigned to record fluorescence transients.

[0106] Postprocessing includes determining the baseline signal before stimulation and comparing it to the response immediately after volatile compound exposure. Increased or decreased neural activity indicates the presence of an agonist or antagonist / inverse agonist, respectively.

[0107] The response can be normalized to a known receptor-agonist pair in the biosensor array, serving as a reference for accurate comparison.

[0108] The present invention also encompasses one or more olfactory signatures. As used herein, an "olfactory signature" refers to a representation of a combinatorial response to volatile compounds by a repertoire of Olfactory Receptors. In one aspect, the representation is a digital representation.

[0109] An olfactory signature usually characterizes said one or more compounds.

[0110] A non-limiting example of an olfactory representation is depicted in Figure 3 A and 3B. Unitec Ref: 1189-B1557 PCT FINAL PAT8401PC00

[0111] In particular aspects, a sequential exposure to volatile compounds can be repeated several times to obtain several successive olfactory signatures of the molecule(s) that can then be statistically processed, for example by averaging or otherwise, to obtain an improved olfactory signature.

[0112] Referring in more detail to the illustrative examples, the Inventors generated transgenic fly lines (e.g. transgenic Drosophila melanogaster) using the GAL4 / UAS system. This method is well-known in the art and is based on the finding that Gal4 binding to UAS sequences activates gene expression (Webster N, Jin JR, Green S, Hollis M, Chambon P. The yeast UAS is a transcriptional enhancer in the presence of the GAL4 trans -activator. Cell. 1988 Jan 29;52(2):169-78). Briefly, the Saccharomyces cerevisiae transactivator GAL4, produced from a driver regulatory cassette, binds as a homodimer to a 17-bp (CGG-Nu -CCG) upstream activating sequence (UAS) on the target transgene cassette.

[0113] The Inventors inserted a GAL4 cassette into the coding sequence of several OlfR on chromosome II, which were then combined through crossing over. Additionally, a UAS- GCaMP transgene was inserted into chromosome II and positioned in cis by crossing over.

[0114] These modified flies were then mated with transgenic flies carrying a UAS-ORx on chromosome III. This breeding produced fully homozygous transgenic fly lines with these mutations that were used in different calcium imaging assays.

[0115] The transgenic fly line stocks were then kept in controlled incubators at a temperature of 25±1°C with a 12: 12 hour light-dark cycle. The flies were bred in glass tubes using a standard medium composed of agar, yeast, cornmeal, fructose, and propionic acid.

[0116] For imaging studies, flies were collected within the specific age range of three to seven days after the emergence of the fly from the pupa. To immobilize the flies for imaging, CO2 pads were employed to induce temporary sedation. Once under sedation, the flies' heads, antennas or maxillary palps were carefully removed using a scalpel. These severed parts were then placed on a nutritious and hydrating gel within a petri dish for observation.

[0117] The invention also relates to a device comprising i) a biosensor, plurality of biosensors, biosensor array, and / or kits of the invention, ii) means for detecting and recording reporter's signal. Unitec Ref: 1189-B1557 PCT FINAL PAT8401PC00

[0118] The present invention provides a method for associating a volatile molecule or mixture of volatile compounds with a combinatorial response pattern of OlfR, the method comprising the steps of: a) providing a biosensor array of the invention, b) contacting the biosensor array with a volatile molecule or mixture of volatile compounds, c) simultaneously detecting the neuronal activity in parallel in each individual biosensor upon exposure to the volatile molecule or mixture of volatile compounds, whereby a change of the neuronal activity in an individual biosensor compared to the baseline indicates a response of the exogenous OlfR expressed in said biosensor to the volatile molecule or mixture of volatile compounds, and d) determining the combinatorial response pattern of OlfRs associated with said volatile molecule or mixture of volatile compounds.

[0119] In one aspect, the invention provides a method for associating a volatile molecule or mixture of volatile compounds with a combinatorial response pattern of OlfRs, comprising detecting the neuronal activity in parallel in each individual biosensor upon a single or more exposure to the volatile molecule or mixture of volatile compounds, whereby a change of the neuronal activity in an individual biosensor compared to the baseline indicates a response of the exogenous OlfR expressed in said biosensor to the volatile molecule or mixture of volatile compounds.

[0120] The invention further provides a method for associating a volatile molecule or mixture of volatile compounds with a combinatorial response pattern of OlfRs, the method comprising the steps of: a) providing a biosensor array of the invention, b) contacting the biosensor array with a volatile molecule or mixture of volatile compounds, c) detecting the neuronal activity in parallel in each individual biosensor upon exposure to the volatile molecule or mixture of volatile compounds, whereby a change of the neuronal activity in an individual biosensor compared to a baseline indicates a response of the exogenous OlfR expressed in said biosensor to the volatile molecule or mixture of volatile compounds, and Unitec Ref: 1189-B1557 PCT FINAL PAT8401PC00 d) determining the combinatorial response pattern of OlfRs associated with said volatile molecule or mixture of volatile compounds.

[0121] In one aspect, the invention provides a method for associating a volatile molecule or mixture of volatile compounds with a combinatorial response pattern of OlfRs, the method comprising the steps of: a) providing a biosensor array of the invention, b) contacting the biosensor array with a volatile molecule or mixture of volatile compounds, c) detecting the neuronal activity in each individual biosensor upon exposure to the volatile molecule or mixture of volatile compounds, whereby a change of the neuronal activity in an individual biosensor compared to a baseline indicates a response of the exogenous OlfR expressed in said biosensor to the volatile molecule or mixture of volatile compounds, and d) determining the combinatorial response pattern of OlfRs associated with said volatile molecule or mixture of volatile compounds.

[0122] As used herein, the term “contacting” in relation to an olfactory receptor present in the biosensor array and a volatile compound, refers to bringing the volatile compound into spatial and functional proximity under conditions suitable to permit an interaction with the olfactory receptor, such as binding, recognition, or activation, between the receptor and the volatile compound. Such contacting may be achieved by any suitable means. The contacting encompasses both transient and sustained exposures sufficient to allow a detectable response or interaction between the olfactory receptor and the volatile compound.

[0123] As used herein, the term “baseline” refers to a reference signal that represents the level of response, activity, or output of the present detection system (biosensor array comprising OlfR(s)) in the absence of a specific stimulus or under defined control conditions. The baseline signal may correspond, for example, to the signal measured prior to exposure to a test compound (a volatile molecule or mixture of volatile compounds). Unless otherwise indicated, the baseline signal serves as a comparative or reference value against which changes in signal intensity (of the neuronal activity in an individual biosensor) upon contacting with a test substance, such as a volatile compound, are determined.

[0124] As used herein, the term “compound” is intended to encompass a molecule as such, including any of its stereochemical forms (e.g., enantiomers, diastereomers, or geometric isomers), Unitec Ref: 1189-B1557 PCT FINAL PAT8401PC00 tautomers, and where applicable, salts, solvates, or hydrates thereof. Thus, reference to a compound shall be understood as referring to a discrete chemical entity defined by a particular molecular structure, i.e. a molecule, unless the context clearly indicates otherwise.

[0125] In one aspect, the detection of the neuronal activity is performed simultaneously in parallel in each individual biosensor of the biosensor array upon exposure to the volatile molecule or mixture of volatile compounds. In other words, the neuronal activity can be detected in each individual biosensor at the same time.

[0126] In one aspect, the detection of the neuronal activity is performed in a sequential or consecutive manner. In other words, the neuronal activity can be detected in each individual biosensor one after the other.

[0127] In one aspect, the biosensor array is repeatedly exposed to same or distinct volatile compounds or mixtures of volatile compounds.

[0128] In one aspect, the neuronal activity is measured by parallel imaging of the calcium flux in each biosensor of the biosensor array.

[0129] In one aspect, the volatile compounds or mixtures of volatile compounds are categorized as a repellent, an attractant, or as compounds modifying reproductive, feeding or aggregation behaviors of the insect.

[0130] Usually, the biosensor array is contacted with a given volatile molecule or mixture of volatile compounds in order to identify the combinatorial response pattern of OlfRs to said volatile molecule or mixture of volatile compounds.

[0131] In one aspect, the biosensor array is sequentially contacted with a plurality of volatile compounds in order to identify volatile compounds or mixtures of volatile compounds that activate a given combinatorial response pattern of OlfRs.

[0132] In one aspect, the biosensor array of the invention comprising at least one OlfR, or a plurality of OlfRs, is firstly contacted with a first volatile compound or mixture of volatile compounds. Then, the same biosensor array of the invention comprising at least one OlfR, or a plurality of Unitec Ref: 1189-B1557 PCT FINAL PAT8401PC00

[0133] OlfRs, is secondly contacted with a second different volatile compound or mixture of volatile compounds. These sequential or consecutive contact with different volatile compound or mixture of volatile compounds (one volatile compound or mixture of volatile compounds after the other) allow the inventors to assess which OlfR(s) are responsive to which volatile compound(s).

[0134] The invention also relates to methods of modulating the behavior of insects / animals comprising distributing into the environment, onto an animal, or onto a surface, object and / or fabric a composition or compound of the invention.

[0135] The invention also relates to a surface, object and / or fabric comprising i) a composition or compound of the invention or ii) coated with a composition or compound of the invention.

[0136] Non-limiting examples of a surface, object and / or fabric are selected from the group comprising plant, fungi, soil, wall, floor, dispenser device, window, furniture, clothing item and packaging material.

[0137] The invention also relates to a method of attracting insects / animals comprising distributing into the environment, onto an animal, or onto a surface the composition or compound of the invention.

[0138] The invention also relates to a method of protecting a plant, seed and / or object from the attack of an animal, e.g. an insect, comprising treating said plant, seed and / or object with a composition or compound of the invention or an agricultural composition thereof.

[0139] The invention also relates to a composition or an agricultural composition. Preferably, the composition or agricultural composition is in the form of a spray, gel, foam, patch, powder, solid, sponge, tape, vapor, paste, tincture, lotion, cream or ointment.

[0140] Suitable forms may be a spray or an aerosol form. The aerosol form may use a liquid or a gas as a propellant. These include, for example, conventional propellant gases required for spray cans, such as propane, butane, dimethyl ether, CO2, or halogenated lower alkyl gases (for example, halogenated Cl -4 alkyls), and mixtures of two or more thereof. In particular, the composition according to the present invention is preferably formulated such that it can be sprayed directly in an area of infestation, it can be bound to a solid support or encapsulated in a time release material. Unitec Ref: 1189-B1557 PCT FINAL PAT8401PC00

[0141] The invention also relates to a method for identifying volatile compounds that modulate the behavior of an insect, comprising the steps of: a) determining the combinatorial response pattern of OlfRs of the insect to a volatile molecule or mixture of volatile compounds that is known to modulate the behavior of the insect in the desired manner as according to the method described herein, and b) identifying volatile compounds or mixtures of volatile compounds that activate the same or a fraction of the combinatorial response pattern of OlfRs as identified under step a).

[0142] The invention also provides a method for the detection of the presence, absence and / or amount of volatile molecule or mixture of volatile compounds in a test sample or a biological sample compared to a reference sample comprising the steps of: a) determining the combinatorial response pattern of OlfRs to the volatile molecule or mixture of volatile compounds in the test sample or biological sample, b) comparing the combinatorial response pattern of OlfRs to the volatile molecule or mixture of volatile compounds in the test sample or biological sample and the reference sample, whereby a different combinatorial response pattern reflects the presence, absence and / or amount of the volatile molecule or mixture of volatile compounds in the test sample or biological sample compared to the reference sample.

[0143] In one aspect, one or more biological sample(s) is / are compared to one or more reference sample(s).

[0144] In one aspect, an additional step of determining the combinatorial response pattern of OlfRs to the volatile molecule or mixture of volatile compounds in the reference samples is performed either during (sequentially with the other steps) or previously of after the method for the detection of the presence, absence and / or amount of volatile molecule or mixture of volatile compounds in a test sample or a biological sample compared to reference samples.

[0145] In one aspect, the invention provides a method for the detection of the presence, absence and / or amount of a volatile molecule or a mixture of volatile compounds in a test sample or biological sample compared to a control sample comprising the steps of: Unitec Ref: 1189-B1557 PCT FINAL PAT8401PC00 a) determining the combinatorial response pattern of OlfRs to the volatile molecule or mixture of volatile compounds in the test sample, b) determining the combinatorial response pattern of OlfRs to the volatile molecule or mixture of volatile compounds in the control sample, c) comparing the combinatorial response pattern of OlfRs to the volatile molecule or mixture of volatile compounds in the test sample and the control sample, whereby a different combinatorial response pattern reflects the presence, absence and / or amount of the volatile molecule or mixture of volatile compounds in the test sample compared to the control sample.

[0146] The invention also relates to one or more volatile compounds or mixtures of volatile compounds identified according to any one of a method described herein.

[0147] The invention further relates to the use of methods and biosensor arrays of the invention to identify OlfR combinatorial response patterns in pest insect species in response to volatile compounds such as pheromones, kairomones, apneumones, allomones, or infochemicals. In one aspect these volatile compounds trigger behaviors such as repellence, attraction, oviposition, feeding, or mating. Manipulating these behaviors in pest insects could help prevent pest damage and nuisance.

[0148] The invention also relates to the screening of large sets of synthetic volatile compounds for their ability to activate OlfRs from a pest insect repertoire. Based on this screen, combinations of synthetic volatile compounds are selected so that taken together they mimic the OlfR combinatorial activation pattern of volatile compounds triggering specific behaviors. These synthetic blends are selected to provide cost-effective, more efficient, and environmentally friendly alternatives to natural volatile compounds for pest control.

[0149] For example, synthetic volatile compound blends identified by using the biosensor array could attract pests to traps, repel them from crops, induce oviposition in traps, mask food odors, or disrupt mating behaviors.

[0150] The set of OlfRs activated by volatile compounds can trigger behaviors through combinatorial activation (multiple OlfRs) or individual activation (single OlfRs). Unitec Ref: 1189-B1557 PCT FINAL PAT8401PC00

[0151] The sensitivity of OlfRs that trigger innate behaviors is often narrowly tuned, preventing activation out of context. This is often the case for pheromone receptors that are specifically tuned to respond to a given pheromone. The method and biosensor of the invention could allow for the identification of such receptors by identifying the receptors that are narrowly tuned, i.e., responding to only a few different volatile compounds.

[0152] The use of the method and biosensor array of the invention can generate a response matrix of entire OlfR repertoires to various volatile compounds. By linking OlfRs to specific behaviors such response matrices can allow to select volatile compounds that generate specific effects.

[0153] The use of the method and biosensor array of the invention can allow to find antagonists blocking the activation of OlfRs triggering specific behaviors in insects. This could for example be useful to block the activity of OlfRs that are responsive to food odors.

[0154] The use of the method and biosensor array of the invention can also allow the identification of volatile compounds able to trigger the innate approach of predators of pest insects (auxiliaries beneficial for the crop).

[0155] The invention further relates to the use of the biosensor array, of the invention to detect pathologies or disease (e.g. cancer) through the detection of volatile compounds (e.g. volatile biomarkers).

[0156] Numerous studies have demonstrated that trained animals can detect diseases, such as cancers, metabolic and systemic diseases, infectious diseases, and neurological and psychiatric conditions, often with an efficiency that matches or surpasses current diagnostic tools. The volatile compounds associated with the diseases can be present in breath, sweat, urine, saliva, blood, and other bodily fluids. The success of these animals serves as a proof of concept that volatile compounds can be used to screen for pathologies; however, large-scale population screening based on trained animals is very difficult to achieve. Therefore, the present invention also allows to screen individuals affected by diseases and based on a disease-specific olfactory signature.

[0157] Following the collection of biological samples form patients with known pathologies, either obtained directly or from previously collected material, the volatile compounds of these samples can be tested on the biosensor array. OlfRs that respond differentially to pathological versus healthy states can be determined by exposing the biosensor to these volatiles and Unitec Ref: 1189-B1557 PCT FINAL PAT8401PC00 evaluating the resulting combinatorial activation patterns. Once disease-specific OlfRs have been determined, biological samples form patients with unknown, suspected or to-be- confirmed pathologies, can be analysed using the same approach. The combinatorial activation pattern of the previously identified OlfRs is then used to determine whether the sample corresponds to a specific health condition or to a non-pathological (healthy) state.

[0158] The invention further relates to one more recombinant nucleic acid(s) encoding at least one exogeneous OlfR and a genetically encoded neuronal activity reporter selected from the group comprising a genetically encoded calcium indicator (GECI), and a genetically encoded voltage indicator (GEVI).

[0159] The invention further relates to an olfactory signature of a test sample containing at least one volatile compound obtained, or obtainable, by a method of the invention.

[0160] The invention also provides a method for diagnosing and / or prognosing a disease in a subject comprising the steps of: a) providing a biosensor array according to the invention, b) contacting the biosensor array with a biological sample derived from the subject, c) detecting the neuronal activity in parallel in each individual biosensor of the biosensor array upon exposure to the volatile molecule or mixture of volatile compounds of the biological sample of step b), whereby a change of the neuronal activity in an individual biosensor compared to a baseline indicates a response of the exogenous OlfR expressed in said biosensor to a volatile molecule or mixture of volatile compounds, d) determining the combinatorial response pattern of OlfRs associated with said volatile molecule or mixture of volatile compounds of the biological sample, e) comparing the combinatorial response pattern of OlfRs to the volatile molecule or mixture of volatile compounds of the biological sample to a reference sample, wherein a differential combinatorial response pattern of OlfRs to the volatile molecule or mixture of volatile compounds of the biological sample relative to the corresponding combinatorial response pattern of OlfRs to the volatile molecule or mixture of volatile compounds of the reference sample is indicative of a diagnosis and / or a prognosis of said disease in the subject. Unitec Ref: 1189-B1557 PCT FINAL PAT8401PC00

[0161] In one aspect, the invention provides a method for screening for and / or diagnosing and / or prognosing a disease in a subject comprising the steps of: a) providing a biosensor array according to the invention, b) contacting the biosensor array with volatile compounds / molecules emanating from a biological sample derived from the subject, c) detecting the neuronal activity in parallel in each individual biosensor of the biosensor array upon exposure to the volatile molecule or mixture of volatile compounds of the biological sample of step b), whereby a change of the neuronal activity in an individual biosensor compared to a baseline indicates a response of the exogenous OlfR expressed in said biosensor to a volatile molecule or mixture of volatile compounds, d) determining the combinatorial response pattern of OlfRs associated with said volatile molecule or mixture of volatile compounds of the biological sample, e) comparing the combinatorial response pattern of OlfRs to the volatile molecule or mixture of volatile compounds of the biological sample to the combinatorial response patterns of reference samples from sick and healthy subjects, wherein the combinatorial response pattern of OlfRs to the volatile molecule or mixture of volatile compounds of the biological sample is more similar to the corresponding combinatorial response patterns of OlfRs to the volatile molecule or mixture of volatile compounds of the reference samples from sick subjects than the combinatorial response patterns from healthy subjects, is indicative of an increased probability and / or diagnosis and / or prognosis of said disease in the subject.

[0162] As used herein, the term "corresponding" in the expression "corresponding combinatorial response pattern of OlfRs" refers to the comparison of the combinatorial response pattern of OlfRs to the volatile molecule or mixture of volatile compounds of the biological sample versus the OlfR combinatorial response to the volatile molecule or mixture of volatile compounds of reference samples, wherein the response pattern of the same OlfRs are compared between the biological sample and the reference samples.

[0163] In one aspect, the reference sample is a reference biological sample (such as a positive or negative control sample, a group of reference samples, a reference value and a previously tested sample). Unitec Ref: 1189-B1557 PCT FINAL PAT8401PC00

[0164] The invention also provides a method for screening for, diagnosing and / or prognosing a disease in a subject comprising the steps of: a) providing a biosensor array of the invention, b) contacting the biosensor array with volatile compounds / molecules emanating from a biological sample derived from the subject, c) detecting the neuronal activity in each individual biosensor of the biosensor array upon exposure to the biological sample of step b), whereby a change of the neuronal activity in an individual biosensor compared to a baseline indicates a response of the exogenous OlfR expressed in said biosensor to a volatile molecule or mixture of volatile compounds, d) determining the response pattern of at least one OlfR associated with said volatile molecule or mixture of volatile compounds of the biological sample, e) comparing the (combinatorial) response pattern of at least one OlfR to the volatile molecule or mixture of volatile compounds of the biological sample to the (combinatorial) response patterns of reference samples from sick and healthy subjects, wherein the combinatorial response pattern of OlfRs to the volatile molecule or mixture of volatile compounds of the biological sample is more similar to the corresponding combinatorial response patterns of OlfRs to the volatile molecule or mixture of volatile compounds of the reference samples from sick subjects than the combinatorial response patterns from healthy subjects, is indicative of an increased probability and / or diagnosis and / or a prognosis of said disease in the subject.

[0165] In one aspect, the invention provides a method for screening for, diagnosing and / or prognosing a disease in a subject, wherein a differential response pattern of at least one OlfR to the volatile molecule or mixture of volatile compounds of the biological sample relative to the corresponding response pattern of the at least same OlfR to the volatile molecule or mixture of volatile compounds of the reference sample is indicative of a diagnosis and / or a prognosis of said disease in the subject.

[0166] As used herein the terms "subject" or "patient" or "individual", are well -recognized in the art, and are used interchangeably herein to refer to a mammal, including, but not limited to a dog, cat, rat, mouse, monkey, cow, horse, goat, sheep, pig, camel, and, most preferably, a human. In some cases, the subject is a subject in need of treatment or a subject with a disease or disorder or condition. However, in other aspects, the subject can be a normal subject (not sick). The Unitec Ref: 1189-B1557 PCT FINAL PAT8401PC00 term does not denote a particular age or sex. Thus, adult and newborn subjects, whether male or female, are intended to be covered. Preferably, the subject is a human, most preferably a human patient having a potential disease, more preferably the potential disease is a cancer.

[0167] As used herein, the term “contacting” in relation to the contact of a biological sample derived from a subject with the biosensor array, refers to bringing the volatile molecule or mixture of volatile compounds (comprised in / forming the biological sample) into spatial and functional proximity under conditions suitable to permit an interaction with the olfactory receptor of the the biosensor array, such as binding, recognition, or activation, between the olfactory receptor and the volatile compound. Such contacting may be achieved by any suitable means. The contacting encompasses both transient and sustained exposures sufficient to allow a detectable response or interaction between the olfactory receptor and the volatile compound.

[0168] In one aspect, the invention provides a method for screening and / or diagnosing and / or prognosing a disease in a subject, wherein the biosensor array comprises at least one OlfR which is contacted sequentially, with the biological sample, and with the reference samples.

[0169] In one aspect, the invention provides a method for screening for and / or diagnosing and / or prognosing a disease in a subject, wherein the biosensor array comprises at least one OlfR which is contacted sequentially, firstly with the reference sample(s), and secondly with the biological sample(s). In one aspect, the biosensor array comprises at least one OlfR which is contacted sequentially, firstly with the biological sample(s), and secondly with the reference sample(s).

[0170] In one aspect, the invention provides a method for screening for and or diagnosing and / or prognosing a disease in a subject, wherein the biosensor array comprises at least 2 OlfRs, at least 3 OlfRs, at least 4 OlfRs, at least 5 OlfRs, at least 6 OlfRs, at least 7 OlfRs, at least 8 OlfRs, at least 9 OlfRs, at least 10 OlfRs, at least 11 OlfRs, at least 12 OlfRs, at least 13 OlfRs, at least 14 OlfRs, at least 15 OlfRs, at least 16 OlfRs, at least 17 OlfRs, at least 18 OlfRs, at least 19 OlfRs, at least 20 OlfRs, at least 25 OlfRs, at least 30 OlfRs, at least 40 OlfRs, at least 50 OlfRs, at least 60 OlfRs, at least 70 OlfRs, at least 80 OlfRs, at least 90 OlfRs, at least 100 or more OlfRs, wherein the biosensor array is contacted sequentially, firstly with reference samples, and secondly with a biological sample, or wherein the biosensor array is contacted sequentially, with a biological sample, and then with reference samples. Unitec Ref: 1189-B1557 PCT FINAL PAT8401PC00

[0171] In one aspect, the biological sample derived from the subject comprises a volatile molecule or mixture of volatile compounds. In one aspect, an additional step of heating the biological sample (derived from the subject) may be required such that it increases volatility by, for example, accelerating the desorption of the volatile molecule or mixture of volatile compounds present in the biological sample. In one aspect, the volatile molecule or mixture of volatile compounds present in the biological sample naturally migrate into the gas phase above the sample.

[0172] In one aspect, the invention also provides a method for screening for and / or diagnosing and / or prognosing a disease in a subject, wherein the detection of the neuronal activity is performed in parallel in each individual biosensor of the biosensor array upon exposure to the biological sample.

[0173] In one aspect, the invention also provides a method for screening for and / or diagnosing and / or prognosing a disease in a subject, wherein the differential response pattern of at least one OlfR to the volatile molecule or mixture of volatile compounds of the biological sample relative to the corresponding response pattern of the at least same OlfR to the volatile molecule or mixture of volatile compounds of the reference sample corresponds to a level of activation or a level of inhibition of the at least one OlfR.

[0174] In one aspect, when a differential response pattern of at least one OlfR to the volatile molecule or mixture of volatile compounds of the biological sample relative to the corresponding response pattern of the at least same OlfR to the volatile molecule or mixture of volatile compounds of the reference samples is observed, a level of activation or a level of inhibition of the at least one OlfR can be associated to the differential response pattern, which can be associated with and / or indicative of a diagnosis and / or a prognosis of said disease in the subject.

[0175] In one aspect, one OlfR may show a differential response pattern between the biological sample and the reference samples, which corresponds to a level of activation, whereas another different OlfR may show a differential response pattern between the biological sample and the reference sample, which corresponds to a level of inhibition.

[0176] In one aspect, an OlfR may show no differential response pattern between the biological sample and the reference samples, which corresponds to an OlfR not specific for diagnosing and / or prognosing a disease or a condition in a subject. Unitec Ref: 1189-B1557 PCT FINAL PAT8401PC00

[0177] In one aspect, the invention also provides a method for screening for and / or diagnosing and / or prognosing a disease in a subject, wherein the disease is selected from an inflammatory disease, a cancer, an autoimmune disease, a neurological disease, an infectious disease and a metabolic disease, or a combination thereof.

[0178] As used herein, the expression “screening for and / or diagnosing and / or prognosing a disease in a subject” refers to the method using the biosensor array, or use of the biosensor array of the invention for the identification, determination, assessment, or prediction of the presence, absence, stage, progression, or likelihood of development of a disease, disorder, or condition in a subject. The terms “screening for and / or diagnosing and / or prognosing a disease in a subject” encompasses the evaluation of one or more biological, clinical, or molecular parameters that are indicative of a pathological or pre-pathological state, or of a predisposition or risk associated with the occurrence or progression of such a disease or condition. The term “screening” includes determining whether a subject currently is more likely than a random individual to be affected by a disease or condition, as well as classifying the disease or condition according to its type, severity, or stage. The term “diagnosing” includes determining whether a subject currently has, or has had, a disease or condition, as well as classifying the disease or condition according to its type, severity, or stage. The term “prognosing” includes predicting or estimating the probable course, outcome, or recurrence of a disease or condition, or assessing a subject’s susceptibility or risk of developing said disease or condition in the future.

[0179] In one aspect, the invention also provides a method for screening for and / or diagnosing and / or prognosing a disease in a subject, wherein the cancer is selected from the group comprising breast cancer, lung cancer, colorectal cancer, prostate cancer, gastric cancer, liver cancer, cervical cancer, thyroid cancer, bladder cancer, non-Hodgkin lymphoma, skin cancer, kidney cancer, pancreatic cancer, ovarian cancer, esophageal cancer, leukemia, endometrial cancer, oral cavity cancer, brain and central nervous system cancers, and multiple myeloma, or a combination thereof.

[0180] In one aspect, the invention also provides a method for screening for and / or diagnosing and / or prognosing a disease in a subject, wherein the inflammatory disease is selected from the group comprising osteoarthritis, asthma, chronic obstructive pulmonary disease (COPD), gout, Unitec Ref: 1189-B1557 PCT FINAL PAT8401PC00 psoriasis, eczema (atopic dermatitis), allergic rhinitis, chronic sinusitis, sarcoidosis, inflammatory bowel disease (Crohn’s disease and ulcerative colitis), atherosclerosis, endometriosis, pancreatitis, non-autoimmune hepatitis, myocarditis, obesity-related chronic inflammation, sepsis, periodontitis, chronic prostatitis, tendonitis, and bursitis, or a combination thereof.

[0181] In one aspect, the invention also provides a method for screening for and / or diagnosing and / or prognosing a disease in a subject, wherein the autoimmune disease is selected from the group comprising rheumatoid arthritis, systemic lupus erythematosus (lupus), type 1 diabetes, multiple sclerosis, Hashimoto’s thyroiditis, Graves’ disease, celiac disease, Sjogren’s syndrome, myasthenia gravis, scleroderma, autoimmune hepatitis, vitiligo, Addison’s disease, pernicious anemia, primary biliary cholangitis, Guillain-Barre syndrome, dermatomyositis, autoimmune hemolytic anemia, antiphospholipid syndrome, and autoimmune uveitis, or a combination thereof.

[0182] In one aspect, the invention also provides a method for screening for / and diagnosing and / or prognosing a disease in a subject, wherein the neurological disease is selected from the group comprising Alzheimer’s disease, Parkinson’s disease, multiple sclerosis, epilepsy, amyotrophic lateral sclerosis (ALS), Huntington’s disease, stroke, peripheral neuropathy, cerebral palsy, traumatic brain injury, cluster headache, Bell’s palsy, Guillain-Barre syndrome, meningitis (neurological manifestation), encephalitis, spinal muscular atrophy, dystonia and trigeminal neuralgia, or a combination thereof.

[0183] In one aspect, the invention also provides a method for screening for / and diagnosing and / or prognosing a disease in a subject, wherein the infectious disease is selected from the group comprising influenza, COVID- 19, tuberculosis, malaria, HIV / AIDS, hepatitis B, hepatitis C, dengue fever, measles, cholera, typhoid fever, syphilis, gonorrhea, human papillomavirus (HPV) infection, Lyme disease, Ebola virus disease, rabies, tetanus, diphtheria, and shingles (herpes zoster), or a combination thereof.

[0184] In one aspect, the invention also provides a method for screening for and / or diagnosing and / or prognosing a disease in a subject, wherein the metabolic disease is selected from the group Unitec Ref: 1189-B1557 PCT FINAL PAT8401PC00 comprising type 2 diabetes, obesity, metabolic syndrome, hyperthyroidism, hypothyroidism, phenylketonuria (PKU), Gaucher disease, Wilson’s disease, hemochromatosis, glycogen storage disease, galactosemia, mitochondrial disease, Cushing’s syndrome, Addison’s disease, familial hypercholesterolemia, gout, acromegaly, congenital adrenal hyperplasia, lipodystrophy, and hypoglycemia, or a combination thereof.

[0185] In one aspect, the invention also provides a method for screening for and / or diagnosing and / or prognosing a disease in a subject, wherein the biological sample is selected from the group comprising exhaled breath, breath, exudates, skin emanations or sweat, underarm sweat, urine, saliva, feces, blood, nasal secretions, nasal mucus, tongue coating or oral biofilm, sebum, foot sweat, scalp or hair, earwax (cerumen), scent glands, tears, milk, vaginal secretions, semen, placenta and amniotic fluid, or a combination thereof.

[0186] In one aspect, the biological sample derived from the subject comprises a volatile molecule or mixture of volatile compounds. In one aspect, an additional step of heating the biological sample (derived from the subject) may be required such that it activates the desorption of the volatile molecule or mixture of volatile compounds present in the biological sample. In one aspect, the volatile molecule or mixture of volatile compounds present in the biological sample naturally migrate into the gas phase above the sample.

[0187] In one aspect, the invention also provides a method for screening for and / or diagnosing and / or prognosing a disease in a subject, wherein the reference sample is selected from the group comprising a control sample, a group of reference samples, a reference value and a previously tested sample, or a combination thereof.

[0188] As used herein, the expression “biological sample” refers to any sample derived from a subject. The biological sample may be obtained from a subject who is affected by or at risk of developing a disease or condition, or from a subject not affected by the disease or condition. The status of the biological sample (diseased or non-diseased) is to be determined, confirmed, or monitored using the methods of the present invention. Unitec Ref: 1189-B1557 PCT FINAL PAT8401PC00

[0189] As used herein, the term “test sample” refers to a material or composition from which volatile compounds are extracted and analysed in order to use and study a volatile molecule or mixture of volatile compounds in the methods of the present invention.

[0190] As used herein, the term “VOC” refers to volatile organic compound(s).

[0191] In one aspect, the test sample is an extract derived from the biological sample.

[0192] The terms “reference sample” and “control sample” are used interchangeably herein and refer to a biological, synthetic, or previously characterized sample that provides a reference value or profile against which one or more parameters of the biological sample are compared. The reference or control sample may be derived from:

[0193] (i) one or more subjects known to be free of the disease or condition (representing a normal or baseline physiological state);

[0194] (ii) one or more subjects known to have the disease or condition (representing a defined pathological state); or

[0195] (iii) a standardized or artificial preparation containing a known quantity, activity, or expression level of one or more biomarkers or analytes of interest or known volatile compounds or VOCs or mixtures of volatile compounds.

[0196] Comparison of the biological sample with the reference or control sample may facilitate the diagnosis, prognosis, risk assessment, or monitoring of the disease or condition in the subject. Unless otherwise indicated, the reference or control sample encompasses negative controls, positive controls, and calibration samples, as appropriate for the analytical or diagnostic method employed.

[0197] In one aspect, the invention also provides a method for screening for and / or diagnosing and / or prognosing a disease in a subject, wherein the biosensor array comprises at least 2 different OlfRs, at least 3 different OlfRs, at least 4 different OlfRs, at least 5 different OlfRs, at least 6 different OlfRs, at least 7 different OlfRs, at least 8 different OlfRs, at least 9 different OlfRs, at least 10 different OlfRs, at least 11 different OlfRs, at least 12 different OlfRs, at least 13 different OlfRs, at least 14 different OlfRs, at least 15 different OlfRs, at least 16 different OlfRs, at least 17 different OlfRs, at least 18 different OlfRs, at least 19 different OlfRs, at least 20 different OlfRs, at least 25 different OlfRs, at least 30 different OlfRs, at least 40 different OlfRs, at least 50 different OlfRs, at least 60 different OlfRs, at least 70 different Unitec Ref: 1189-B1557 PCT FINAL PAT8401PC00

[0198] OlfRs, at least 80 different OlfRs, at least 90 different OlfRs, at least 100 or more different OlfRs.

[0199] In one aspect, the invention also provides a method for screening for and / or diagnosing and / or prognosing a disease in a subject, wherein the biosensor array comprises at least 2 identical OlfRs, at least 3 identical OlfRs, at least 4 identical OlfRs, at least 5 identical OlfRs, at least 6 identical OlfRs, at least 7 identical OlfRs, at least 8 identical OlfRs, at least 9 identical OlfRs, at least 10 identical OlfRs, at least 11 identical OlfRs, at least 12 identical OlfRs, at least 13 identical OlfRs, at least 14 identical OlfRs, at least 15 identical OlfRs, at least 16 identical OlfRs, at least 17 identical OlfRs, at least 18 identical OlfRs, at least 19 identical OlfRs, at least 20 identical OlfRs, at least 25 identical OlfRs, at least 30 identical OlfRs, at least 40 identical OlfRs, at least 50 identical OlfRs, at least 60 identical OlfRs, at least 70 identical OlfRs, at least 80 identical OlfRs, at least 90 identical OlfRs, at least 100 or more identical OlfRs.

[0200] In one aspect, the invention also provides a method for screening for and / or diagnosing and / or prognosing a disease in a subject, wherein each different OlfR is present in at least two identical exemplars in the biosensor array.

[0201] In one aspect, the invention also provides a method for screening for and / or diagnosing and / or prognosing a disease in a subject, wherein each different OlfR is present in at least two identical biosensors in the biosensor array.

[0202] In one aspect, each different OlfR is present in at least two, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100 identical exemplars in the biosensor array.

[0203] In one aspect, the invention also provides a method for screening for and / or diagnosing and / or prognosing a disease in a subject, wherein the biosensor array is contacted firstly with the reference sample and secondly with the biological sample, or firstly with the biological sample and secondly with the reference sample.

[0204] In one aspect, the invention also provides a method for screening for and / or diagnosing and / or prognosing a disease in a subject, wherein the biosensor array is repeatedly exposed to a biological sample and / or to a reference sample. Unitec Ref: 1189-B1557 PCT FINAL PAT8401PC00

[0205] In one aspect, the invention also provides a method for screening for and / or diagnosing and / or prognosing a disease in a subject, wherein the biosensor array is repeatedly exposed to one or more biological sample and / or to one or more reference sample.

[0206] In one aspect, the invention also provides a method for diagnosing and / or prognosing a disease in a subject, further comprising treating the subject based upon the identified differential combinatorial response pattern of OlfRs indicative of a diagnosis and / or a prognosis of a disease in the subject.

[0207] In one aspect, the invention also provides a method for screening for and / or diagnosing and / or prognosing a disease in a subject, further comprising treating the subject based upon the identified differential combinatorial response pattern of OlfRs indicative of an increased probability of and / or a diagnosis and / or a prognosis of a disease in the subject.

[0208] In one aspect, the invention also provides a method for screening for and / or diagnosing and / or prognosing a disease in a subject, further comprising treating the subject based upon the identified differential response pattern of at least one OlfR indicative of an increased probability of and / or a diagnosis and / or a prognosis of a disease or condition in the subject.

[0209] In one aspect, the invention also provides a method for screening for and / or diagnosing and / or prognosing a disease or a condition in a subject, further comprising monitoring the relapse or evolution of the disease or the efficacy of a treatment.

[0210] In one aspect, the invention also provides a method for identifying Olfactory Receptor(s) (OlfR(s)) whose response pattern is / are specific to a volatile molecule or mixture of volatile compounds associated with diseases, comprising the step of: identifying OlfR(s) having a differential combinatorial response patterns upon exposure to the volatile molecule or mixture of volatile compounds of the biological sample relative to the corresponding combinatorial response patterns of OlfRs to the volatile molecule or mixture of volatile compounds of reference samples, wherein the differential combinatorial response pattern of OlfRs is indicative of an increased probability of and / or a diagnosis and / or a prognosis of a disease in the subject. Unitec Ref: 1189-B1557 PCT FINAL PAT8401PC00

[0211] In one aspect, the invention provides the means to identify at least one OlfR whose response pattern is / are specific to a volatile molecule or mixture of volatile compounds associated with disease(s). The number of identified "disease-specific" OlfR is not limited by the invention.

[0212] In one aspect, at least 1 OlfR, at least 2 OlfRs, at least 3 OlfRs, at least 4 OlfRs, at least 5 OlfRs, at least 6 OlfRs, at least 7 OlfRs, at least 8 OlfRs, at least 9 OlfRs, at least 10 OlfRs, at least 11 OlfRs, at least 12 OlfRs, at least 13 OlfRs, at least 14 OlfRs, at least 15 OlfRs, at least 16 OlfRs, at least 17 OlfRs, at least 18 OlfRs, at least 19 OlfRs, at least 20 OlfRs, at least 30 OlfRs, at least 40 OlfRs, at least 50 OlfRs, at least 60 OlfRs, at least 70 OlfRs, at least 80 OlfRs, at least 90 OlfRs, at least 100 OlfRs or more OlfRs are identified as "disease-specific" OlfRs using the method of the invention.

[0213] Ine one aspect, the identified "disease-specific" OlfRs are used in the method for screening for and / or diagnosing and / or prognosing a disease in a subject according to the invention.

[0214] The invention further provides a method of treatment of a disease in a subject, comprising the steps of: a) identifying OlfRs with a differential combinatorial response pattern indicative of a diagnosis and / or a prognosis of a disease in the subject according to the invention, b) treating the subject based upon the differential combinatorial response pattern of the identified OlfRs indicative of a diagnosis and / or a prognosis of a disease in the subject.

[0215] In one aspect, the invention also provides a method of treatment of a disease in a subject, comprising the steps of: a) identifying OlfRs with a differential combinatorial response pattern indicative of an increased probability of and / or diagnosis and / or a prognosis of a disease in the subject according to the invention, b) treating the subject based upon the differential combinatorial response pattern of the identified OlfRs indicative of an increased probability of and / or diagnosis and / or a prognosis of a disease in the subject. Unitec Ref: 1189-B1557 PCT FINAL PAT8401PC00

[0216] In one aspect, the invention provides a method comprising administering to the subject a therapeutically effective amount of a treatment composition based upon the diagnosis and / or a prognosis of a disease in the subject. The treatment composition may include one or more active agents selected from small molecules, biologies, nucleic acid-based therapeutics, cellular therapies, or combinations thereof. In some aspects, the treatment may further comprise adjunctive therapies such as immunomodulatory agents, anti-inflammatory agents, antimicrobial compounds, chemotherapeutic agents, metabolic regulators, or neuroprotective compounds. The administration may be carried out via any suitable route, including systemic, parenteral, oral, or local delivery, optionally in combination with controlled-release or targeted- delivery formulations.

[0217] The invention further provides a kit for performing any methods according to the invention, said kit comprising a) the biosensor array of the invention, b) means and / or reagents for determining the combinatorial response pattern of OlfR(s), and / or c) instructions for use.

[0218] In one aspect, the kit of the invention further comprises a reference sample (or a reference value) as defined above.

[0219] In one aspect, the kit of the invention further comprises controls, standards and / or calibrators. In one aspect, the kit of the invention further comprises means for collecting the biological sample from the subject.

[0220] In one aspect, the kit of the invention further comprises one or more containers for compositions contained in the kit. Compositions can be in liquid form or can be lyophilized. Suitable containers for the compositions include, for example, bottles, vials, syringes, and test tubes. Containers can be formed from a variety of materials, including glass or plastic.

[0221] In one aspect, the invention provides the use of the kit of the invention in a method for associating a volatile molecule or mixture of volatile compounds with a combinatorial response pattern of OlfRs of the invention. Unitec Ref: 1189-B1557 PCT FINAL PAT8401PC00

[0222] In one aspect, the invention provides the use of the kit of the invention in a method for identifying volatile compounds that modulate the behavior of an insect according to the invention.

[0223] The invention further provides the use of the kit of the invention in a method for the detection of the presence, absence and / or amount of volatile molecule or mixture of volatile compounds in a test sample or biological sample compared to a reference sample according to the invention.

[0224] The invention further provides the use of the kit of the invention in a method for diagnosing or prognosing a disease in a subject according to the invention.

[0225] In one aspect, the invention further provides the use of the kit of the invention in a method for screening for or diagnosing or prognosing a disease in a subject according to the invention.

[0226] In one aspect, the invention provides the use of the kit of the invention in a method for identifying Olfactory Receptor(s) (OlfR(s)) with disease-associated response pattern according to the invention.

[0227] The invention further provides the use of the kit of the invention in a method of treatment of a disease in a subject of the invention.

[0228] The invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications without departing from the spirit or essential characteristics thereof. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features. The present disclosure is therefore to be considered as in all aspects illustrated and not restrictive, the scope of the invention being indicated by the appended Claims, and all changes which come within the meaning and range of equivalency are intended to be embraced therein. Various references are cited throughout this Specification, each of which is incorporated herein by reference in its entirety. The foregoing description will be more fully understood with reference to the following Examples. Unitec Ref: 1189-B1557 PCT FINAL PAT8401PC00

[0229] Unitec Ref: 1189-B1557 PCT FINAL PAT8401PC00

[0230] EXAMPLES

[0231] Material & Methods

[0232] Transgenic fly stock and transgenesis

[0233] The Drosophila melanogaster stocks were kept in incubators at a controlled temperature of 25±1°C with a 12: 12 hour light-dark cycle. The flies were bred in glass tubes using a standard medium composed of agar, yeast, cornmeal, fructose, and propionic acid. For imaging studies, flies were collected within the specific age range of three to seven days post-emergence. To immobilize the flies for imaging, CO2 pads were employed to induce temporary sedation. Once under sedation, the flies' heads or antennas were removed. These severed parts were then placed on a nutritious gel within a petri dish.

[0234] For the empty neuron approach, ORxGAL4, UASGCaMP flies were generated (see also infra). Transgenes containing OlfRs from different insect species (D. melanogaster, H. armigera,) were generated (20xUASORx), and the corresponding flies were crossed with the ORxGAL4, UASGCaMP flies.

[0235] Preparation of volatile compounds

[0236] Volatile compound solutions were prepared through serial dilutions using distilled water and / or DMSO or other solvents when needed. Initially, most volatile compounds were tested at a 1000X dilution.

[0237] Exposure to volatile compounds

[0238] An Aurora Scientific 220A olfactometer was utilized to administer volatile compound pulses. During purging phases, blank stimuli, and intervals between pulses, only purified air was flowed at a steady rate of 380 seem. During the release of volatile compounds, air carrying the volatile compounds from the vial pathway mixes with clean air at a 1 :20 ratio, yielding 19 seem of volatile compound-laden air and 361 seem of dilution air, thus maintaining a consistent total airflow throughout the experiment.

[0239] Volatile compound samples were loaded into Eppendorf tubes, which were placed in amber vials incorporated into the olfactometer's tubing system. A custom software program managed the timing of volatile compound pulses, which consisted of 30 seconds of volatile compound- charged air building up at the final valve, followed by a 1 -second burst of volatile compounds, Unitec Ref: 1189-B1557 PCT FINAL PAT8401PC00

[0240] 60 seconds of accumulation, another 1 -second burst, 60 seconds of further accumulation, a 30- second purge with clean air, and finally, a 1 -second blank burst, culminating in a total sequence duration of 184 seconds. This sequence was replicated for each volatile compound test, with water controls implemented between trials to check for any possible system contamination. After the stimulation concluded, all valves shut, and clean air was used to purge the trial plate, ensuring no residual volatile compounds were left behind.

[0241] Calcium Imaging

[0242] Volatile compound trials were captured using aZeiss SteREO Lumar vl2 microscope to record fluorescence signals. Recordings were made at a frame rate of 6.6 frames per second. Subsequently, regions of interest (ROIs) were defined around the antennas. The ZEN program was used to capture the fluorescent responses.

[0243] Statistical Analysis

[0244] The raw data were analyzed using an R-coded pipeline. A rolling AF / F method was implemented, utilizing the average of the previous 25 values for calculations. Response peaks were identified and synchronized using autocorrelation algorithms by evaluating the fluorescence slope. In instances where no responses were detected, the timing of the stimulation was automatically aligned with their theoretical timing. Subsequently, a rolling Z-Score was calculated based on the standard deviation and mean of the preceding 150 frames. The highest Z-Score value, indicative of the response peak, was extracted for creating comparative boxplots. For each volatile compound trial, plots of normalized traces, aligned general Z-Score traces, response Z-Score variations, and boxplots for each experimental condition were produced. Additionally, boxplot summaries on a consistent scale for all conditions were also created to provide a comprehensive overview of the experiment.

[0245] Urine Collection and Treatment

[0246] Urine samples were obtained from the remains of experimental stocks used for another experiment. Six-week-old male C57BL / 6 mice were injected intraperitoneally with lipopolysaccharide (LPS; 1 mg / kg in 200 uL saline) to induce systemic inflammation, while control animals received an equivalent volume (1 mL) of sterile saline. Urine was collected beginning 4 hours after injection(s) and over a 3-hours period. Samples were pooled from five individuals per condition, and each tested sample consisted of 1 mL of pooled urine. Unitec Ref: 1189-B1557 PCT FINAL PAT8401PC00

[0247] 1 - Results

[0248] We generated, via CRISPR / Cas9 targeting, transgenic Drosophila melanogaster flies with a GAL4 cassette replacing the coding sequences (CDS) of olfactory receptor genes, DmOR22a, DmOR59b, and DmOR42b, all located on chromosome II and expressed in the medio-proximal segments of the antennae. This approach, using empty neuron populations located in an exposed restricted area of the antenna, allows an efficient signal collection. In parallel, we inserted a 20xUAS-GCaMP transgene into two attP sites on chromosome II via PhiC31 integrase recombination. We then used chromosomal crossing overs to combine the two 20xUAS-GCaMP on the same chromosome, together with either of the GAL4 lines. The CDSs of OlfRs tested were cloned into 20x-UAS expression vectors and integrated into different attP2 sites on chromosome III. The 20xUAS-ORx transgenic lines were then crossed with the transgenic lines carrying either ORGal4 and the two 20UAS-GCaMP to generate fully homozygous Drosophila melanogaster flies (Fig. la).

[0249] While some insect species antennae can be maintained alive and recorded for extended periods of time after being cut, the small Drosophila antennae, when isolated, show a rapid dampening of signal in response to volatile compounds, possibly due to a combination of desiccation, nutrient deficiency, and homeostasis imbalance. We reasoned that if we could maintain Drosophila antennae or heads alive and functional after separation from the rest of the body, it would be possible to pack many samples for parallel recording via transcuticular calcium imaging. We developed a novel an air-gel organotypic culture approach where the dissected antennae or heads are placed on a nutritive gel matrix, allowing the upper part of the cultured tissue to be accessible for stimulation with volatile compounds. We tested several gel compositions, including in which the medium was composed of sensilla ringer, artificial hemolymph solution, Drosophila Ringer, and Schneider's medium. For long-term culture, Schneider's medium provided the best results and was selected for subsequent experiments. Using this approach, we were able to simultaneously record responses to volatile compounds from at least 100 Drosophila heads or antennae under a microscope (Fig. lb). This setup represents a breakthrough compared to traditional methods that handle a maximum of four live flies at a time, allowing for the assessment of entire insect OlfR repertoires in single experiments. Unitec Ref: 1189-B1557 PCT FINAL PAT8401PC00

[0250] All the following experiments are derived from parallel recordings of numerous Drosophila heads or antennae. Volatile compound-induced responses largely exceeded noise, and the GCaMP fluorescence showed minimal bleaching after multiple stimulations or long volatile compound exposures. We considered five standard deviations above the mean as a threshold corresponding to a significant response.

[0251] The shape of the calcium imaging traces in response to volatile compounds varied as well as the time they took to return to baseline, depending on the OlfR and the identity and concentration of the tested ligands. For example, DmOR22a, when activated with ethyl hexanoate, showed sustained responses, while activation with other tested volatiles did not induce the same surge pattern (Fig.2a). The strength of the observed responses was dosedependent (Fig.2b), and the OlfR sensitivities to specific ligands were similar to those reported by in vivo techniques elsewhere.

[0252] The stability of this setup was highlighted by its ability to yield consistent responses despite numerous volatile compound stimulations (Fig.2c). Remarkably, we observed that Drosophila heads or antennae placed on the nutrient gel matrix could remain alive for up to at least six days. This is particularly practical for testing large sets of volatile compounds over extended time periods (Fig.2d).

[0253] Example 2 - Results

[0254] We selected 15 Drosophila OlfRs and tested them against 15 volatile compounds, thus assessing 225 DmOR-ligand pairs. These response patterns are reproducible and similar to known receptor-ligands pairs reported in the literature (Fig.3a). However, when compared to single-sensillum recordings, our method shows a more bimodal distribution between responding and non-responding OlfRs. This difference unlikely arises from our unability to detect responses below some thresholds, as we observed dose responses of OlfRs to volatile compounds until high levels of dilution. Thus, the clearer distinction between responsive and non-responsive OlfR-volatile compound pairs is likely due to reduced noise arising from signal amplification and / or the filtering out of weakly responding OlfRs when measuring calcium concentrations at the level of the olfactory sensory neurons’ soma. Confirming our method applicability beyond Drosophila, we transgenically introduced HarmOR40 and HarmOR43 OlfRs from the pest butterfly Helicoverpa armigera into the Drosophila empty neuron system, and observed responses to their known ligands, respectively, geranyl acetate and Unitec Ref: 1189-B1557 PCT FINAL PAT8401PC00 phenylacetaldehyde (Fig.3b). We confirmed that HarmOR40 and HarmOR43 are narrowly tuned. We also confirmed that specific OlfRs of Drosophila are less narrowly tuned than others. We also observed that some volatile compounds activated a large proportion of the Drosophila OlfRs, in particular compounds linked to plants. For example, l-octen-3-ol, which is produced by various plant species, triggers responses in many OlfRs of Drosophila.

[0255] 3 - Results

[0256] In addition to odorant receptors, ionotropic receptors are also able to bind volatile compounds in insects. To assess our ability to express and functionally test this type of chemoreceptor in our system, we co-expressed DmIR84a with its co-receptor DmIR8a via a 2A self-cleaving peptide (Fig.4a).

[0257] We observed a response of the corresponding transgenic Drosophila to the expected DmIR84a / DmIR8a ligand phenylacetaldehyde. The response levels were within the same range as those observed with Drosophila and Helicoverpa armigera ORs. The return to the baseline following volatile compound-induced activation took several minutes. This is a known peculiarity of some specific OlfR-ligand interactions.

[0258] 4 - Results

[0259] The control of pest insects by volatile compounds would greatly benefit from inverse agonists or antagonists as these would be able to mask naturally occurring odors, such as those associated with food or inducing oviposition. To test the ability of our method to identify such volatile compounds, we exposed OR59b to a known OR59b inhibitor, linalool, and observed a decrease in baseline fluorescence.

[0260] Moreover, we were able to reverse the ethyl acetate activation of OR59b (a known agonist), by concomitantly exposing it to linalool (Fig.4b). This represents a proof of principle that our device is able to help identifying compounds that could potentially mask volatile compounds that may naturally attract pest species.

[0261] 5 - Results

[0262] A multi-species panel of ORs was exposed to samples from rodents exhibiting an inflammatory state induced by intraperitoneal (IP) injection of LPS (lipopolysaccharide), mimicking a bacterial infection, and to IP saline controls. The tested biological samples included pooled urine headspace. In the results shown in Figure 5 (a, b), only the ORs from Delia platura (a Unitec Ref: 1189-B1557 PCT FINAL PAT8401PC00 dipteran pest, “bean fly”) selected for their differential response between sample types are shown. Four D. platura ORs allow for reproducible discrimination between sick and healthy individuals. The approach is adaptable to other type of samples (e.g., exhaled breath, exudates, skin emanations, insect headspace) and to alternative OR panels, depending on the organisms and conditions of interest.

[0263] Example 5 illustrates the capacity to screen and distinguish subjects on the basis of modulations in their VOC profiles. Biological samples may be obtained from urine, exhaled breath, sweat, saliva, blood, feces, exudates / secretions, skin headspace, cage / bedding headspace (for rodents), insect headspace, and related biological media. Reliable identification of affected individuals establishes status and informs subsequent management, particularly in human subjects, while simultaneously mapping the activated ORs.

[0264] References

[0265] Dobritsa AA, van der Goes van Naters W, Warr CG, Steinbrecht RA, Carlson JR. Integrating the molecular and cellular basis of odor coding in the Drosophila antenna. Neuron. 2003 Mar 6;37(5): 827-41.

[0266] Mariette J, Noel A, Louis T, Montagne N, Chertemps T, Jacquin-Joly E, Marion-Poll F, Sandoz JC. Transcuticular calcium imaging as a tool for the functional study of insect odorant receptors. Front Mol Neurosci. 2023 Aug 14; 16: 1182361.

Claims

Unitec Ref: 1189-B1557 PCT FINAL PAT8401PC00CLAIMS1. A biosensor array comprising a plurality of biosensors, wherein each biosensor comprises a severed transgenic Drosophila head or olfactory sensory organ (antenna and / or maxillary palp) of said transgenic Drosophila positioned so that at least one antenna and / or maxillary palp is in contact with air, and the head or olfactory sensory organ is in contact with a nutritious and hydrating matrix, and wherein the cells of the antenna and / or the maxillary palp lack endogenous Olfactory Receptors (OlfRs) and co-express at least one exogenous insect OlfR and a genetically encoded neuronal activity reporter.

2. The biosensor array of claim 1, wherein the plurality of biosensors comprises at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100 or more drosophila heads or olfactory sensory organs.

3. The biosensor array of any one of the preceding claims, wherein the biosensor array comprises an entire OlfR repertoire of at least one insect.

4. The biosensor array of any one of the preceding claims, wherein the nutritious and hydrating matrix is provided in the form of a gelatinous matrix, a thin layer of liquid, or a liquid in a porous matrix and comprises balanced salt solutions, amino acids, vitamins, buffers, serum or serum replacements, hydrolysates and growth factors, or a combination thereof.

5. The biosensor array of any one of the preceding claims, wherein the genetically encoded neuronal activity reporter is selected from the group comprising a genetically encoded calcium indicator (GECI), and a genetically encoded voltage indicator (GEVI).

6. The biosensor array of claim 5, wherein the GECI is selected from the group comprising GCaMP, R-GECOls, GEM-GECO, and Chameleon, a variant or a combination thereof.Unitec Ref: 1189-B1557 PCT FINAL PAT8401PC007. The biosensor array of any one of the preceding claims, wherein the at least one exogeneous OlfR belongs to at least one insect selected from the group comprising an insect that i) is harmful to agricultural crops, ii) presents a safety risk for humans or animals and / or iii) causes a nuisance for humans.

8. A method for associating a volatile molecule or mixture of volatile compounds with a combinatorial response pattern of OlfRs, the method comprising the steps of: a) providing a biosensor array of any one of claims 1 to 7, b) contacting the biosensor array with a volatile molecule or mixture of volatile compounds, c) detecting the neuronal activity in parallel in each individual biosensor upon exposure to the volatile molecule or mixture of volatile compounds, whereby a change of the neuronal activity in an individual biosensor compared to a baseline indicates a response of the exogenous OlfR expressed in said biosensor to the volatile molecule or mixture of volatile compounds, and d) determining the combinatorial response pattern of OlfRs associated with said volatile molecule or mixture of volatile compounds.

9. The method of claim 8, wherein the biosensor array is repeatedly exposed to same or distinct volatile compounds or mixtures of volatile compounds.

10. The method of any one of claims 8 and 9, wherein the neuronal activity is measured by parallel imaging of the calcium flux in each biosensor of the biosensor array.

11. The method of any one of claims 8 to 10, wherein, the volatile compounds or mixtures of volatile compounds are categorized as a repellent, an attractant, or as compounds modifying reproductive, feeding or aggregation behaviors of the insect.

12. The method of any one of claims 8 to 11, wherein the biosensor array is contacted with a given volatile molecule or mixture of volatile compounds in order to identify the combinatorial response pattern of OlfRs to said volatile molecule or mixture of volatile compounds.Unitec Ref: 1189-B1557 PCT FINAL PAT8401PC0013. The method of any one of claims 8 to 11, wherein the biosensor array is sequentially contacted with a plurality of volatile compounds in order to identify volatile compounds or mixtures of volatile compounds that activate a given combinatorial response pattern of OlfRs.

14. The method of any one of claims 8 to 11 for identifying volatile compounds that modulate the behavior of an insect, comprising the steps of: a) determining the combinatorial response pattern of OlfRs of the insect to a volatile molecule or mixture of volatile compounds that is known to modulate the behavior of the insect in the desired manner, and b) identifying volatile compounds or mixtures of volatile compounds that activate the same or a fraction of the combinatorial response pattern of OlfRs as identified under step a).

15. The method of any one of claims 8 to 11 for the detection of the presence, absence and / or amount of volatile molecule or mixture of volatile compounds in a test sample or a biological sample compared to a reference sample comprising the steps of: a) determining the combinatorial response pattern of OlfRs to the volatile molecule or mixture of volatile compounds in the test sample or biological sample, b) comparing the combinatorial response pattern of OlfRs to the volatile molecule or mixture of volatile compounds in the test sample or biological sample and the reference sample, whereby a different combinatorial response pattern reflects the presence, absence and / or amount of the volatile molecule or mixture of volatile compounds in the test sample or biological sample compared to the reference sample.

16. One or more volatile compounds or mixtures of volatile compounds identified according to the method of any one of claims 8 to 14.

17. A method for diagnosing and / or prognosing a disease in a subject comprising the steps of:Unitec Ref: 1189-B1557 PCT FINAL PAT8401PC00 a) providing a biosensor array according to claims 1 to 6, b) contacting the biosensor array with a biological sample derived from the subject, c) detecting the neuronal activity in parallel in each individual biosensor of the biosensor array upon exposure to the volatile molecule or mixture of volatile compounds of the biological sample of step b), whereby a change of the neuronal activity in an individual biosensor compared to a baseline indicates a response of the exogenous OlfR expressed in said biosensor to a volatile molecule or mixture of volatile compounds, d) determining the combinatorial response pattern of OlfRs associated with said volatile molecule or mixture of volatile compounds of the biological sample, e) comparing the combinatorial response pattern of OlfRs to the volatile molecule or mixture of volatile compounds of the biological sample to a reference sample, wherein a differential combinatorial response pattern of OlfRs to the volatile molecule or mixture of volatile compounds of the biological sample relative to the corresponding combinatorial response pattern of OlfRs to the volatile molecule or mixture of volatile compounds of the reference sample is indicative of a diagnosis and / or a prognosis of said disease in the subject.

18. The method of claim 17, wherein the disease is selected from an inflammatory disease, cancer, an autoimmune disease, a neurological disease, an infectious disease and a metabolic disease, or a combination thereof.

19. The method of any one of claims 17 and 18, wherein the cancer is selected from the group comprising breast cancer, lung cancer, colorectal cancer, prostate cancer, gastric cancer, liver cancer, cervical cancer, thyroid cancer, bladder cancer, non-Hodgkin lymphoma, skin cancer, kidney cancer, pancreatic cancer, ovarian cancer, esophageal cancer, leukemia, endometrial cancer, oral cavity cancer, brain and central nervous system cancers, and multiple myeloma, or a combination thereof.

20. The method of any one of claims 17 to 19, wherein the inflammatory disease is selected from the group comprising osteoarthritis, asthma, chronic obstructive pulmonary disease (COPD), gout, psoriasis, eczema (atopic dermatitis), allergic rhinitis, chronic sinusitis, sarcoidosis, inflammatory bowel disease (Crohn’s disease and ulcerative colitis), atherosclerosis, endometriosis, pancreatitis, non-autoimmune hepatitis, myocarditis, obesity-Unitec Ref: 1189-B1557 PCT FINAL PAT8401PC00 related chronic inflammation, sepsis, periodontitis, chronic prostatitis, tendonitis, and bursitis, or a combination thereof.

21. The method of any one of claims 17 to 20, wherein the autoimmune disease is selected from the group comprising rheumatoid arthritis, systemic lupus erythematosus (lupus), type 1 diabetes, multiple sclerosis, Hashimoto’s thyroiditis, Graves’ disease, celiac disease, Sjogren’s syndrome, myasthenia gravis, scleroderma, autoimmune hepatitis, vitiligo, Addison’s disease, pernicious anemia, primary biliary cholangitis, Guillain-Barre syndrome, dermatomyositis, autoimmune hemolytic anemia, antiphospholipid syndrome, and autoimmune uveitis, or a combination thereof.

22. The method of any one of claims 17 to 21, wherein the neurological disease is selected from the group comprising Alzheimer’s disease, Parkinson’s disease, multiple sclerosis, epilepsy, amyotrophic lateral sclerosis (ALS), Huntington’s disease, stroke, peripheral neuropathy, cerebral palsy, traumatic brain injury, cluster headache, Bell’s palsy, Guillain- Barre syndrome, meningitis (neurological manifestation), encephalitis, spinal muscular atrophy, dystonia and trigeminal neuralgia, or a combination thereof.

23. The method of any one of claims 17 to 22, wherein the infectious disease is selected from the group comprising influenza, COVID-19, tuberculosis, malaria, HIV / AIDS, hepatitis B, hepatitis C, dengue fever, measles, cholera, typhoid fever, syphilis, gonorrhea, human papillomavirus (HPV) infection, Lyme disease, Ebola virus disease, rabies, tetanus, diphtheria, and shingles (herpes zoster), or a combination thereof.

24. The method of any one of claims 17 to 23, wherein the metabolic disease is selected from the group comprising type 2 diabetes, obesity, metabolic syndrome, hyperthyroidism, hypothyroidism, phenylketonuria (PKU), Gaucher disease, Wilson’s disease, hemochromatosis, glycogen storage disease, galactosemia, mitochondrial disease, Cushing’s syndrome, Addison’s disease, familial hypercholesterolemia, gout, acromegaly, congenital adrenal hyperplasia, lipodystrophy, and hypoglycemia, or a combination thereof.

25. The method of claim 15 or the method of any one of claims 17 to 24, wherein the biological sample is selected from the group comprising exhaled breath, breath, exudates, skin emanations or sweat, underarm sweat, urine, saliva, feces, blood, nasal secretions, nasal mucus,Unitec Ref: 1189-B1557 PCT FINAL PAT8401PC00 tongue coating or oral biofilm, sebum, foot sweat, scalp or hair, earwax (cerumen), scent glands, tears, milk, vaginal secretions, semen, placenta and amniotic fluid, or a combination thereof.

26. The method of claim 15 or the method of any one of claims 17 to 25, wherein the reference sample is selected from the group comprising a control sample, a group of reference samples, a reference value and a previously tested sample, or a combination thereof.

27. The method of any one of claims 17 to 26, wherein the biosensor array comprises at least 2 different OlfRs, at least 3 different OlfRs, at least 4 different OlfRs, at least 5 different OlfRs, at least 6 different OlfRs, at least 7 different OlfRs, at least 8 different OlfRs, at least 9 different OlfRs, at least 10 different OlfRs, at least 11 different OlfRs, at least 12 different OlfRs, at least 13 different OlfRs, at least 14 different OlfRs, at least 15 different OlfRs, at least 16 different OlfRs, at least 17 different OlfRs, at least 18 different OlfRs, at least 19 different OlfRs, at least 20 different OlfRs, at least 25 different OlfRs, at least 30 different OlfRs, at least 40 different OlfRs, at least 50 different OlfRs, at least 60 different OlfRs, at least 70 different OlfRs, at least 80 different OlfRs, at least 90 different OlfRs, at least 100 or more different OlfRs.

28. The method of any one of claims 17 to 27, wherein each different OlfR is present in at least two identical biosensors in the biosensor array.

29. The method of any one of claims 17 to 28, wherein the biosensor array is contacted firstly with the reference sample and secondly with the biological sample, or firstly with the biological sample and secondly with the reference sample.

30. The method of any one of claims 17 to 29, wherein the biosensor array is repeatedly exposed to a biological sample and / or to a reference sample.

31. The method of any one of claims 17 to 30, further comprising treating the subj ect based upon the identified differential combinatorial response pattern of OlfRs indicative of a diagnosis and / or a prognosis of a disease in the subject.Unitec Ref: 1189-B1557 PCT FINAL PAT8401PC0032. The method of any one of claims 17 to 31, further comprising monitoring the relapse or evolution of the disease or the efficacy of a treatment.

33. The method of any one of claims 17 to 32 for identifying OlfR(s) whose response pattern is / are specific to a volatile molecule or mixture of volatile compounds associated with diseases, comprising the step of: identifying OlfR(s) having a differential combinatorial response pattern upon exposure to the volatile molecule or mixture of volatile compounds of the biological sample relative to the corresponding combinatorial response pattern of OlfRs to the volatile molecule or mixture of volatile compounds of the reference sample, wherein the differential combinatorial response pattern of OlfRs is indicative of a diagnosis and / or a prognosis of a disease in the subject.

34. A method of treatment of a disease in a subject, comprising the steps of: a) identifying OlfRs with a differential combinatorial response pattern indicative of a diagnosis and / or a prognosis of a disease in the subject according to claims 17 to 33, b) treating the subject based upon the differential combinatorial response pattern of the identified OlfRs indicative of a diagnosis and / or a prognosis of a disease in the subject.

35. A kit for performing the methods according to any one of claims 8 to 15 and 17 to 34, said kit comprising a) the biosensor array of any one of claims 1 to 7, b) means and / or reagents for determining the combinatorial response pattern of OlfR(s), and / or c) instructions for use.

36. Use of the kit of claim 35 in a method for the detection of the presence, absence and / or amount of volatile molecule or mixture of volatile compounds in a test sample or biological sample compared to a reference sample according to claim 15.

37. Use of the kit of claim 35 in a method for diagnosing or prognosing a disease in a subject of any one of claims 17 to 32.Unitec Ref: 1189-B1557 PCT FINAL PAT8401PC0038. Use of the kit of claim 35 in a method of treatment of a disease in a subject of claim 34.

Images