Method and system for detecting odor molecules

Abstract

The present application relates to the technical field of odor molecule detection, and particularly relates to an odor molecule detection method and system. The present application is characterized in that a diaphragm with a hole is installed in an electrolytic cell to divide the electrolytic cell into a forward chamber cavity and a reverse chamber cavity; a circle of hydrophobic organic solvent layer is arranged along the outer periphery of the hole; electrolyte is added into the forward chamber cavity and the reverse chamber cavity, and a phospholipid solution is added above the electrolyte to form a phospholipid bilayer on the hole; insect olfactory receptor protein is added into the forward chamber cavity, the insect olfactory receptor protein is inserted into the phospholipid bilayer to form a closed channel; a sample containing a target odor molecule is added into the forward chamber cavity, the insect olfactory receptor protein recognizes the target odor molecule and combines with the target odor molecule to form an open channel, under the action of an electric field force, electrolyte ions pass through the open channel to generate a current signal, which is used for target odor molecule detection. The present application solves the problem that the existing electrochemical sensor needs to be chemically modified and has low sensitivity and selectivity.

Description

Technical Field

[0001] This invention relates to the field of odor molecule detection technology, specifically to an odor molecule detection method and system. Background Technology

[0002] Currently, odor detection technologies mainly include traditional gas sensor technology, biosensor technology, electronic nose technology, and gas chromatography-mass spectrometry (GC-MS). These odor detection technologies differ in their detection principles, application scenarios, and performance characteristics, and also face technical challenges related to stability, selectivity, detection limit, response time, equipment size, and power consumption.

[0003] Traditional gas sensors detect odors based on different transducers and conventional materials, such as field-effect transistors, quartz crystal microbalances, surface acoustic waves, surface plasmon resonance, optically addressed potential sensors, microelectrode arrays, and fluorescence sensors. These gas sensors are widely used, but they have certain limitations in terms of sensitivity and selectivity, and also suffer from problems such as short lifespan, high cost, and poor stability.

[0004] CN113686937A discloses an electrochemical sensor and detection method based on an odor-binding protein. The electrochemical sensor is constructed by modifying pretreated porous alumina nanopores with tris(2-carboxyethyl)phosphine hydrochloride aqueous solution, activating them with EDC / NHS solution, adjusting the pH, adding human odor-binding protein, mixing the solution by shaking, incubating at low temperature, and then rinsing to obtain the electrochemical sensor. The human odor-binding protein is hOBP 2B. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride is abbreviated as EDC; N-hydroxysuccinimide is abbreviated as N-Hydroxysuccinimide. This electrochemical sensor can be applied to detect fruit aroma compounds. The specific detection method involves dissolving target molecules, such as vanillin, lauric acid, and ethyl acetate, in a methanol solution of a specific volume fraction to prepare a series of concentration solutions. Using a self-made three-electrode system, an electrochemical method is employed for detection, and the concentration of the target substance is determined by the current-time curve. Alternatively, the electrochemical sensor and sample can be placed in a sealed container, and the current signal reflects the content of odor compounds in the sample. However, existing electrochemical sensors require chemical modification and have limitations in sensitivity and selectivity. Furthermore, pretreatment steps are necessary before detection, which are time-consuming and costly. Summary of the Invention

[0005] To address the problem that existing electrochemical sensors require chemical modification and have low sensitivity and selectivity, this invention provides a method and system for detecting odor molecules.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows.

[0007] The first aspect of this invention provides a method for detecting odor molecules, comprising the following steps:

[0008] A porous membrane is installed inside an electrolytic cell to divide the cell into a cis-chamber and an anti-chamber. A hydrophobic organic solvent layer is arranged around the periphery of the pores. Electrolyte is added to the cis-chamber and anti-chamber, and then a phospholipid solution is added above the electrolyte. As the electrolyte level in the cis-chamber and anti-chamber increases, a phospholipid bilayer forms on the pores. Insect olfactory receptor proteins are added to the cis-chamber, and these proteins insert into the phospholipid bilayer to form closed channels. A sample containing target odor molecules is added to the cis-chamber, and the insect olfactory receptor proteins recognize and bind to the target odor molecules, forming open channels. Under the action of an electric field, electrolyte ions pass through the open channels, generating a current signal. The characteristic current blocking signal parameter in the current signal is used for the detection of target odor molecules.

[0009] In existing technologies, the solid nanopores used require prior modification. Human odor-binding proteins need to be incubated at low temperatures for 2 hours, followed by rinsing, before the sensor can be obtained within the solid nanopores. In contrast, the channel of this invention is a biological nanochannel, obtained by adding insect olfactory receptors to an electrolyte and then inserting them into a phospholipid bilayer. In the odor molecule detection method of this invention, the insect olfactory receptor protein requires no pretreatment or chemical modification, and the detection method is rapid, accurate, sensitive, and has a relatively low detection limit.

[0010] This invention identifies a variety of odor molecules through the distributed hydrophobic interactions of insect olfactory receptor proteins and specifically binds to them, enabling highly sensitive detection of low concentrations of target odor molecules.

[0011] Preferably, the insect olfactory receptor protein is MhOR5; MhOR5 has four transmembrane helices, which form a hydrophobic binding pocket for recognizing and binding to target odor molecules; MhOR5 has a trigger helix for regulating the closed or open state of the channel; the method for forming an open channel is: when the hydrophobic binding pocket binds to the target odor molecule, the trigger helix expands outward to regulate the channel from the closed state to the open state.

[0012] Preferably, the characteristic current blocking signal parameters are generated in the current signal as the target odor molecule binds to and dissociates from the receptor protein; the characteristic current blocking signal parameters are a dataset of blocking current amplitude and blocking time.

[0013] A preferred method for detecting target odor molecules is:

[0014] The dataset of blocking current amplitude and blocking time in the current signal is compared with the dataset of blocking current amplitude and blocking time corresponding to different concentrations of target odor molecules for the detection of target odor molecules.

[0015] A further preferred method for detecting target odor molecules is as follows:

[0016] A sample containing the target odor molecule is added to the chamber. Under the action of an electric field, the sample containing the target odor molecule is detected to obtain the current signal of the target odor molecule, as well as the data set of blocking current amplitude and blocking time in the current signal. The data set of blocking current amplitude and blocking time in the current signal is compared with the data set of blocking current amplitude and blocking time corresponding to target odor molecules of different concentration gradients to obtain the concentration of the target odor molecule.

[0017] A further preferred method for obtaining the dataset of blocking current amplitude and blocking time corresponding to target odor molecules at different concentration gradients is as follows:

[0018] Samples containing target odor molecules of different concentration gradients are added to the chamber. Under the action of an electric field, the samples containing target odor molecules of different concentration gradients are detected to obtain a series of current signals of target odor molecules of different concentration gradients. Based on the series of current signals of target odor molecules of different concentration gradients, the differences in blocking current amplitude and blocking time of the series of current signals are classified to obtain a dataset of blocking current amplitude and blocking time corresponding to target odor molecules of different concentration gradients.

[0019] Preferably, the electric field force is generated under a voltage of -40mV to -90mV. More preferably, the voltage is -40mV to -80mV, such as -40mV, -60mV, -80mV, and -90mV.

[0020] Preferably, the hydrophobic organic solvent layer is a mixture of pentane and hexadecane, and the hexadecane accounts for 1% to 5% of the mass percentage of the mixture.

[0021] Preferably, the electrolyte contains Na + Mg + K + Ca 2+An aqueous solution of 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid; the pH of the electrolyte is 7-8.

[0022] In this invention, the electrolyte contains Na + The concentration was 96 mmol / L, Mg + The concentration was 5 mmol / L, K + The concentration was 2 mmol / L, Ca 2+ The concentration of [unspecified substance] is 0.8 mmol / L, and the concentration of 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid is 5 mmol / L. Preferably, the pH of the electrolyte is 7.4. [The following appears to be unrelated and possibly a separate sentence fragment:] Na + NaCl, Mg + For MgCl2, K + KCl, Ca 2+ It is CaCl2.

[0023] Preferably, the concentration of the phospholipid solution is 10 mg / mL, specifically DPhPC, manufactured by Avanti PolarLipids, USA. In this invention, if the concentration of the phospholipid solution is too high, the protein cannot easily insert and cannot form channels; if the concentration of the phospholipid solution is too low, the phospholipids cannot spread properly and are not easy to form a film.

[0024] DPhPC is an abbreviation for 1,2-Diphytanoyl-sn-glycero-3-phosphocholine. It is a synthetic phosphatidylcholine derivative with the molecular formula C2. 48 H 96 NO8P, molecular weight 846.25.

[0025] Preferably, the number of pores is one, and the diameter of the pore is 120μm to 140μm; the diaphragm is a polytetrafluoroethylene membrane.

[0026] A second aspect of the present invention provides an odor molecule detection system for performing the aforementioned odor molecule detection method; the odor molecule detection system includes: an electrolytic cell, wherein a diaphragm is installed within the electrolytic cell to divide the electrolytic cell into a cis-chamber and an anti-chamber; the diaphragm has pores, and a hydrophobic organic solvent layer is disposed around the outer periphery of the pores; an electrolyte and a phospholipid solution are added to both the cis-chamber and the anti-chamber, and a phospholipid bilayer is formed on the pores as the liquid level of the electrolyte in the cis-chamber and the anti-chamber increases; an insect olfactory receptor protein is inserted into the phospholipid bilayer from one side of the cis-chamber to form a closed channel, for recognizing and binding to target odor molecules, and adjusting the channel to switch from a closed state to an open state; and a data acquisition system connected to the electrolytic cell for generating a current signal containing characteristic current blocking signal parameters for target odor molecule detection.

[0027] Preferably, electrodes are installed in both the orthogonal and anticortical chambers. The data acquisition system includes a microcurrent amplification unit, a digital-to-analog conversion unit, a function generation unit, and a processing unit connected in sequence. The microcurrent amplification unit is connected to both electrodes. The odor molecule detection system also includes a power supply. Both the electrolytic cell and the data acquisition system are connected to the power supply.

[0028] The beneficial effects of this invention are:

[0029] 1. This invention identifies a variety of odor molecules through the distributed hydrophobic interactions of insect olfactory receptor proteins and specifically binds to them, enabling high-sensitivity detection of low concentrations of target odor molecules. This solves the problem that existing electrochemical sensors require chemical modification and have low sensitivity and selectivity.

[0030] 2. The method of the present invention has a detection limit of up to the picomolar level when detecting S-cis-verbenol, α-pinene and (-)-verbenone, and is responsive to a variety of chemical substances, thus having broad chemical adaptability.

[0031] 3. The odor molecule detection method of the present invention broadens the detection range and meets the needs of odor molecule detection in different scenarios. The operation is relatively simple. Compared with other complex odor detection methods, although the operation of the present invention involves multiple steps, the operation is relatively standardized and does not require complicated pretreatment, which reduces the difficulty of operation and detection cost.

[0032] 4. This invention uses nanochannels formed by the MhOR5 olfactory receptor protein as its core. Without modification or labeling, it detects terpene odor molecules by means of the current signal characteristics induced by the binding of a single molecule to the protein, such as the amplitude of the blocking current and the blocking time. The sensitivity reaches the picomolar level and is suitable for the detection of pheromones of agricultural and forestry pests. Attached Figure Description

[0033] Figure 1 This is a schematic diagram of the odor molecule detection system provided in an embodiment of the present invention. (a) is a schematic diagram of the component used to seal the electrolytic cell; (b) is a schematic diagram of the connection structure between the electrolytic cell and the data acquisition system; and (c) is a schematic diagram of the insertion of insect olfactory receptor proteins into the phospholipid bilayer.

[0034] Figure 2 This diagram illustrates two states of the channel in an insect olfactory receptor protein. (a) shows the channel of the insect olfactory receptor protein in the closed state; (b) shows the channel of the insect olfactory receptor protein in the open state after binding to odor molecules.

[0035] Figure 3 The current signal is for S-cis-verbenol at a concentration of 1 pM.

[0036] Figure 4 The current signal is for S-cis-verbenol at a concentration of 10 nM.

[0037] Figure 5 The current signal is for S-cis-verbenol at a concentration of 10 μM.

[0038] Figure 6 The current signal is for α-pinene at a concentration of 10 pM.

[0039] Figure 7 The current signal is for α-pinene at a concentration of 10 nM.

[0040] Figure 8 The current signal is for α-pinene at a concentration of 10 μM.

[0041] Figure 9 This is a schematic diagram showing the change in blocking current amplitude with the concentration of α-pinene.

[0042] Figure 10 This is a schematic diagram of the dataset of blocking current amplitude and blocking time for α-pinene.

[0043] Figure 11 The current signal is for (-)-verbenone at a concentration of 10 pM.

[0044] Figure 12 The current signal is for (-)-verbenone at a concentration of 10 nM.

[0045] Figure 13 The current signal is for (-)-verbenone at a concentration of 10 μM.

[0046] Explanation of reference numerals in the attached figures:

[0047] 10. Electrolytic cell; 11. Diaphragm; 12. Cyclic chamber; 13. Anti-cyclic chamber; 14. Pore; 15. Phospholipid bilayer; 16. Insect olfactory receptor protein; 17. Electrode; 18. Electrolyte inlet; 19. Electrode socket; 20. Data acquisition system; 21. Microcurrent amplification unit; 22. Digital-to-analog conversion unit; 23. Function generation unit; 24. Processing unit; 30. Odor molecule. Detailed Implementation

[0048] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0049] Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0050] The technical solution of the present invention will be further described below through specific embodiments.

[0051] In the following embodiments, unless otherwise specified, the methods described are conventional methods; and unless otherwise specified, the reagents and materials described are commercially available.

[0052] In the following embodiments, the polytetrafluoroethylene membrane is also known as a Telfon membrane.

[0053] HEPES stands for 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid; its structural formula is: In the electrolyte, the HEPES buffer solution is made by dispersing HEPES powder in deionized water and stirring until completely dissolved.

[0054] like Figure 1 An odor molecule detection system includes an electrolytic cell 10, insect olfactory receptor proteins 16, a data acquisition system 20, and a power supply. The data acquisition system 20 is connected to the electrolytic cell 10, and both the electrolytic cell 10 and the data acquisition system 20 are connected to the power supply. This connection is achieved through electrical wires. The data acquisition system 20 generates a current signal containing characteristic current blocking signal parameters for odor molecule detection.

[0055] An electrolytic cell 10 is equipped with a diaphragm 11 to divide the electrolytic cell 10 into a symmetric chamber 12 and an anti-symmetric chamber 13. The diaphragm 11 has pores 14 and a hydrophobic organic solvent layer is arranged around the outer periphery of the pores 14. Electrolyte and phospholipid solution are added to both the symmetric chamber 12 and the anti-symmetric chamber 13. As the liquid level of the electrolyte in the symmetric chamber 12 and the anti-symmetric chamber 13 increases, a phospholipid bilayer 15 is formed on the pores 14.

[0056] In this embodiment of the invention, the diaphragm 11 is a polytetrafluoroethylene membrane; the electrolytic cell 10 is divided into a forward chamber 12 and a reverse chamber 13 by the polytetrafluoroethylene membrane, and a circular hole with a diameter of about 120 μm to 140 μm is prepared on the diaphragm 11 using an electric spark generator.

[0057] In this embodiment of the invention, a pre-perforated Telfon membrane is installed in the center of an electrolytic cell 10, dividing the electrolytic cell 10 into two parts. A mixture of pentane and hexadecane containing 1% hexadecane is added dropwise around the pores 14 using a capillary tube. 700 μL of electrolyte and a small amount of phospholipid solution are added to the cis-chamber 12 and the anti-chamber 13. The electrolyte is prepared by mixing 96 mmol / L NaCl, 2 mmol / L KCl, 0.8 mmol / L CaCl2, 15 mmol / L MgCl2, and 5 mmol / L HEPES, with a pH of 7.4. The concentration of the phospholipid solution is 10 mg / mL. Approximately 800 μL of electrolyte is added to the cis-chamber 12 and the anti-chamber 13 until the liquid level extends beyond the pores 14, forming a stable phospholipid bilayer on the pores 14.

[0058] Insect olfactory receptor protein 16 inserts into the phospholipid bilayer 15 from one side of the cis-chamber 12 to form a closed channel. After recognizing and binding to the target odor molecule 30, it regulates the channel to switch from a closed state to an open state, allowing the cations in the cis-chamber 12 to move to the anti-chamber 13 through the open channel.

[0059] In this embodiment of the invention, the insect olfactory receptor protein is MhOR5. MhOR5 has four transmembrane helices, which form a hydrophobic binding pocket for recognizing and binding to target odor molecules. MhOR5 also has a trigger helix for regulating the closed or open state of the channel. The four transmembrane helices are transmembrane helix S2, transmembrane helix S3, transmembrane helix S4, and transmembrane helix S6; the trigger helix is ​​trigger helix S7.

[0060] Specifically, MhOR5 is added to the cis-chamber chamber 12 of the electrolytic cell 10. MhOR5 is inserted into the phospholipid bilayer 15 from one side of the cis-chamber chamber 12 to form a closed channel. A sample containing the target odor molecule at a certain concentration is added to the cis-chamber chamber 12 of the electrolytic cell 10, stirred evenly, and the current signal corresponding to the target odor molecule is detected at a voltage of -60mV. The transmembrane helices S2, S3, S4, and S6 of MhOR5 form a 15Å deep hydrophobic binding pocket. Through distributed hydrophobic interactions of aromatic and hydrophobic residues, the target odor molecule is recognized. After binding with the target odor molecule, the trigger helical S7 expands outward, switching the channel at the center of MhOR5 from a closed state to an open state, thus allowing ions to pass through. The detection principle is as follows. Figure 2 As shown.

[0061] In this embodiment of the invention, during the detection process, the channel state switches rapidly between open and closed. When a certain period of time has elapsed since the detection began, the channel opening frequency decreases and the channel returns to the closed state.

[0062] In this embodiment of the invention, electrodes 17 are installed in both the orthogonal chamber 12 and the anti-orthogonal chamber 13. The data acquisition system 20 includes a micro-current amplification unit 21, a digital-to-analog conversion unit 22, a function generation unit 23, and a processing unit 24 connected in sequence. The micro-current amplification unit 21 is connected to both electrodes 17. Specifically, both the orthogonal chamber 12 and the anti-orthogonal chamber 13 have an electrolyte inlet 18 and an electrode insertion port 19.

[0063] In this embodiment of the invention, the microcurrent amplification unit 21 is a microcurrent amplifier, the digital-to-analog conversion unit 22 is a digital-to-analog converter, the function generation unit 23 is a function generator, and the processing unit 24 is a computer; the data acquisition system 20 is composed of the microcurrent amplifier, the digital-to-analog converter, the function generator, and the computer, as shown below. Figure 1 As shown, changes in current signals at the picoampere level are captured, amplified, and transmitted to the computer. During the experiment, the Ag / AgCl electrode was connected to the electrolytic cell and the probe of the microcurrent amplifier, and the electrolytic cell was placed in a Faraday cage to reduce noise interference from external electromagnetic waves and vibrations.

[0064] An odor molecule detection method includes the following steps:

[0065] Step 1: Install a porous membrane inside the electrolytic cell to divide the electrolytic cell into a symmetric chamber and an anti-symmetric chamber; arrange a hydrophobic organic solvent layer around the outer periphery of the pores; add electrolyte to the symmetric chamber and the anti-symmetric chamber, and then add a phospholipid solution above the electrolyte. As the liquid level of the electrolyte in the symmetric chamber and the anti-symmetric chamber increases, a phospholipid bilayer is formed on the pores.

[0066] Specifically, the number of holes is one, and the diameter of the hole is 120μm to 140μm; the diaphragm is a polytetrafluoroethylene membrane.

[0067] The hydrophobic organic solvent layer is a mixture of pentane and hexadecane, with hexadecane comprising 1% to 5% of the mixture by mass. For example, the hexadecane may comprise 1%, 2%, 2%, 3%, or 5% of the mixture by mass. The function of the hydrophobic organic solvent layer is to disperse the phospholipids, which helps to form a stable phospholipid bilayer on the pores.

[0068] The electrolyte contains Na. + Mg + K + Ca 2+ An aqueous solution of 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid; the pH of the electrolyte is 7-8. For example, in the electrolyte, Na... + The concentration was 96 mmol / L, Mg + The concentration was 5 mmol / L, K + The concentration was 2 mmol / L, Ca 2+ The concentration of [unspecified substance] is 0.8 mmol / L, and the concentration of 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid is 5 mmol / L. The pH of the electrolyte is 7.4. [The following appears to be unrelated and possibly a separate sentence fragment:] Na + NaCl, Mg + For MgCl2, K + KCl, Ca 2+ It is CaCl2.

[0069] The increase in the electrolyte level in the forward and reverse chambers is achieved by adding electrolyte to the forward and reverse chambers, causing the electrolyte level to extend beyond the orifice.

[0070] Step 2: Insect olfactory receptor protein is added to the pleural chamber, and the insect olfactory receptor protein is inserted into the phospholipid bilayer to form a closed channel.

[0071] Specifically, the insect olfactory receptor protein is MhOR5; MhOR5 has four transmembrane helices, which form a hydrophobic binding pocket for recognizing and binding to target odor molecules; MhOR5 has a trigger helix for regulating the closed or open state of the channel; the method for forming an open channel is: when the hydrophobic binding pocket binds to the target odor molecule, the trigger helix expands outward to regulate the channel from the closed state to the open state.

[0072] Step 3: Add a sample containing the target odor molecule into the pleural chamber. The insect olfactory receptor protein recognizes the target odor molecule and binds to it to form an open channel. Under the action of an electric field, electrolyte ions pass through the open channel to generate a current signal. The characteristic current blocking signal parameter in the current signal is used for the detection of the target odor molecule.

[0073] Specifically, the characteristic current blocking signal parameters are generated in the current signal as the target odor molecule binds to and dissociates from the receptor protein; the characteristic current blocking signal parameters are a dataset of blocking current amplitude and blocking time.

[0074] The method used for the detection of target odor molecules is:

[0075] The dataset of blocking current amplitude and blocking time in the current signal is compared with the dataset of blocking current amplitude and blocking time corresponding to different concentrations of target odor molecules for the detection of target odor molecules.

[0076] Specifically, the method for detecting target odor molecules is as follows:

[0077] A sample containing the target odor molecule is added to the chamber. Under the action of an electric field, the sample containing the target odor molecule is detected to obtain the current signal of the target odor molecule, as well as the data set of blocking current amplitude and blocking time in the current signal. The data set of blocking current amplitude and blocking time in the current signal is compared with the data set of blocking current amplitude and blocking time corresponding to target odor molecules of different concentration gradients to obtain the concentration of the target odor molecule.

[0078] The method for obtaining the dataset of blocking current amplitude and blocking time corresponding to target odor molecules at different concentration gradients is as follows:

[0079] Samples containing target odor molecules of different concentration gradients are added to the chamber. Under the action of an electric field, the samples containing target odor molecules of different concentration gradients are detected to obtain a series of current signals of target odor molecules of different concentration gradients. Based on the series of current signals of target odor molecules of different concentration gradients, the differences in blocking current amplitude and blocking time of the series of current signals are classified to obtain a dataset of blocking current amplitude and blocking time corresponding to target odor molecules of different concentration gradients.

[0080] In this embodiment of the invention, the electric field force is generated under a voltage of -40mV to -90mV. More preferably, the voltage is -40mV to -80mV, such as -40mV, -60mV, -80mV, and -90mV.

[0081] In this embodiment of the invention, a sample containing the target odor molecule is added to the cis-chamber, and a voltage of -60mV is applied across the electrolytic cell. The insect olfactory receptor protein recognizes the target odor molecule and binds to it, forming an open channel. This allows ions in the cis-chamber to move directionally through the open channel to the anti-chamber, generating a current signal containing characteristic current blocking signal parameters for odor molecule detection.

[0082] In embodiments of the present invention, such as Figure 2 The MhOR5 insect receptor has seven helices, designated S1, S2, S3, S4, S5, S6, and S7. The S2, S3, S4, and S6 helices of the MhOR5 insect receptor form a 15 Å deep hydrophobic binding pocket. The bottom and walls of this pocket are composed of aromatic and hydrophobic residues, such as Phe, Tyr, and Trp for aromatics, and Leu, Ile, and Val for hydrophobic residues. Phe represents phenylalanine; Tyr represents tyrosine; Trp represents tryptophan; Leu represents leucine; Ile represents isoleucine; and Val represents valine. The MhOR5 insect receptor recognizes odor molecules through distributed hydrophobic interactions between aromatic and hydrophobic residues. After binding to an odor molecule, the MhOR5 insect receptor triggers the outward expansion of the S7 helix, switching the closed central channel from a closed state to an open state, thereby allowing cations to pass through.

[0083] Following the method described above, target odor molecules, including S-cis-verbenol, α-pinene, and (-)-verbenone, were detected at different concentration gradients. The results are as follows. Figures 3 to 13 As shown, by statistically analyzing a series of current signals between target odor molecules at different concentration gradients, and classifying the differences in blocking current amplitude and blocking time based on these signals, a dataset of blocking current amplitude and blocking time corresponding to target odor molecules at different concentration gradients is obtained. This dataset is then used to detect the concentration of samples containing the target odor molecules. The detection methods for other odor molecules are similar.

[0084] In the same ecosystem, S-cis-verbenol, α-pinene, and (-)-verbenone form a dynamic network through complex chemical transformations and biological regulation. α-pinene is the main monoterpene compound released by coniferous trees such as pine, and as a basic substance, it can be transformed into the other two components through biological or abiotic pathways.

[0085] Example 1

[0086] An odor molecule detection method includes the following steps:

[0087] S1. Using S-cis-verbenol as the target odor molecule, a standard sample containing the target odor molecule is diluted to the required detection concentration to obtain a series of test samples with concentration gradients of the target odor molecule. The specific concentration gradients are 1 pM, 10 nM, and 10 μM.

[0088] S2. A sample of target odor molecules with a series of concentration gradients is added to the chamber. A voltage of -60mV is applied to both sides of the electrolytic cell. Under the action of the electric field, the sample of target odor molecules with a series of concentration gradients is used to detect the target odor molecules, obtaining a series of current signals for the target odor molecules with a series of concentration gradients. Based on the series of current signals for the target odor molecules with a series of concentration gradients, such as... Figures 3-5 The differences in blocking current amplitude and blocking time of a series of current signals are classified to obtain a dataset of blocking current amplitude and blocking time corresponding to target odor molecules with a series of concentration gradients.

[0089] The lowest detection limit of S-cis-verbenol was determined using the above-described odor molecule detection method.

[0090] like Figures 3-5 As shown in the results, the detection limit for S-cis-verbenol is 1 pM.

[0091] Example 2

[0092] An odor molecule detection method includes the following steps:

[0093] S1. Using α-pinene as the target odor molecule, a standard sample containing the target odor molecule is diluted to the required detection concentration to obtain a series of concentration gradients of the target odor molecule in the test sample. The specific concentration gradients are 10 pM, 10 nM, and 10 μM.

[0094] S2. A sample of target odor molecules with a series of concentration gradients is added to the chamber. A voltage of -60mV is applied to both sides of the electrolytic cell. Under the action of the electric field, the sample of target odor molecules with a series of concentration gradients is used to detect the target odor molecules, obtaining a series of current signals for the target odor molecules with a series of concentration gradients. Based on the series of current signals for the target odor molecules with a series of concentration gradients, such as... Figures 6-8 The differences in blocking current amplitude and blocking time of a series of current signals were classified to obtain the variation of blocking current amplitude with α-pinene concentration, such as... Figure 9 As shown, and a dataset of blocking current amplitudes and blocking times corresponding to target odor molecules at a series of concentration gradients, as shown in the figure. Figure 10 As shown.

[0095] The lowest detection limit of α-pinene was determined using the above-described odor molecule detection method.

[0096] like Figures 6-10 As shown in the results, the detection limit for α-pinene is 10 pM.

[0097] Example 3

[0098] An odor molecule detection method includes the following steps:

[0099] S1. Using (-)-vervaline as the target odor molecule, a standard sample containing the target odor molecule is diluted to the required detection concentration to obtain a series of concentration gradients of the target odor molecule in the test sample. The specific concentration gradients are 10 pM, 10 nM, and 10 μM.

[0100] S2. A sample of target odor molecules with a series of concentration gradients is added to the chamber. A voltage of -60mV is applied to both sides of the electrolytic cell. Under the action of the electric field, the sample of target odor molecules with a series of concentration gradients is used to detect the target odor molecules, obtaining a series of current signals for the target odor molecules with a series of concentration gradients. Based on the series of current signals for the target odor molecules with a series of concentration gradients, such as... Figures 11-13 The differences in blocking current amplitude and blocking time of a series of current signals are classified to obtain a dataset of blocking current amplitude and blocking time corresponding to target odor molecules with a series of concentration gradients.

[0101] The lowest detection limit of (-)-verbenone was determined by using the above-described odor molecule detection method.

[0102] like Figures 11-13 As shown in the figure, the detection results indicate that the limit of detection for (-)-vervaline is 10 pM.

[0103] Furthermore, subsequent experiments confirmed that the odor molecule detection method of the above embodiments of the present invention can also be used for the detection of vanillin, lauric acid, and ethyl acetate, and the detection limit can reach the femtogram level. In summary, the odor molecule detection method of the embodiments of the present invention can react to a variety of chemical substances and has broad chemical adaptability.

[0104] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for detecting odor molecules, characterized in that, Includes the following steps: A porous diaphragm is installed inside the electrolytic cell to divide the cell into a common chamber and a reverse chamber; a hydrophobic organic solvent layer is disposed around the outer periphery of the pores; the hydrophobic organic solvent layer is a mixture of pentane and hexadecane, and the hexadecane accounts for 1% to 5% of the mass percentage of the mixture; Electrolyte is added to the orthogonal chamber and the anti-orthogonal chamber, and then a phospholipid solution is added above the electrolyte. As the liquid level of the electrolyte in the orthogonal chamber and the anti-orthogonal chamber increases, a phospholipid bilayer is formed on the pore. Insect olfactory receptor proteins are added to the pleural chamber, and the insect olfactory receptor proteins are inserted into the phospholipid bilayer to form a closed channel; A sample containing the target odor molecule is added to the pleural chamber, and the insect olfactory receptor protein recognizes the target odor molecule and binds to it to form an open channel; Under the influence of an electric field, electrolyte ions pass through the open channel, generating a current signal. The characteristic current blocking signal parameter in the current signal is used to detect the target odor molecule; the target odor molecule is S-cis-verbenol, α-pinene, or (-)-verbenone. The insect olfactory receptor protein is MhOR5; MhOR5 has four transmembrane helices, which form a hydrophobic binding pocket for recognizing and binding to target odor molecules; MhOR5 has a trigger helix for regulating the closed or open state of the channel. The method for forming an open channel is as follows: when the hydrophobic binding pocket binds to the target odor molecule, it triggers the helix to expand outward, thereby adjusting the channel to switch from a closed state to an open state; The characteristic current blocking signal parameters are generated in the current signal as the target odor molecule binds to and dissociates from the receptor protein; the characteristic current blocking signal parameters are a dataset of blocking current amplitude and blocking time. The method used for the detection of target odor molecules is: The dataset of blocking current amplitude and blocking time in the current signal is compared with the dataset of blocking current amplitude and blocking time corresponding to different concentrations of target odor molecules for the detection of target odor molecules.

2. The odor molecule detection method according to claim 1, characterized in that, The electric field force is generated under a voltage of -40mV to -90mV.

3. The odor molecule detection method according to claim 1, characterized in that, The electrolyte contains Na. + Mg + K + Ca 2+ An aqueous solution of 4-(2-hydroxyethyl)azine-1-ethanesulfonic acid; the pH of the electrolyte is 7-8.

4. The odor molecule detection method according to claim 1, characterized in that, The number of holes is one, and the diameter of the hole is 120μm to 140μm; the diaphragm is a polytetrafluoroethylene membrane.

5. An odor molecule detection system, characterized in that, Used to perform the odor molecule detection method according to any one of claims 1 to 4; The odor molecule detection system includes: An electrolytic cell is provided, wherein a diaphragm is installed inside the electrolytic cell to divide the electrolytic cell into a cis-chamber and an anti-chamber; the diaphragm has pores, and a hydrophobic organic solvent layer is disposed around the outer periphery of the pores; an electrolyte and a phospholipid solution are added to both the cis-chamber and the anti-chamber, and as the liquid level of the electrolyte in the cis-chamber and the anti-chamber increases, a phospholipid bilayer is formed on the pores; Insect olfactory receptor proteins are inserted into the phospholipid bilayer from one side of the pleural chamber to form a closed channel. After recognizing and binding to the target odor molecule, the channel is regulated to switch from a closed state to an open state. A data acquisition system, connected to the electrolytic cell, is used to generate a current signal containing characteristic current blocking signal parameters for the detection of target odor molecules.

6. The odor molecule detection system according to claim 5, characterized in that, Electrodes are installed in both the orthogonal and anticortical chambers. The data acquisition system includes a microcurrent amplification unit, a digital-to-analog conversion unit, a function generation unit, and a processing unit connected in sequence. The microcurrent amplification unit is connected to the two electrodes respectively. The odor molecule detection system also includes a power supply, and both the electrolytic cell and the data acquisition system are connected to the power supply.

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