Multi-channel odorant molecule detection sensor, system and application

Abstract

The invention belongs to the technical field of odorant molecule detection, and relates to a multi-channel odorant molecule detection sensor, system and application, the sensor comprises a detection cavity having a common grounding cavity and a plurality of independent detection cavities, the common grounding cavity and the plurality of independent detection cavities are used for accommodating a detection buffer solution carrying odorant molecules to be detected, a detection channel is arranged between each independent detection cavity and the public grounding cavity; a lipid bilayer is loaded on each detection channel, and olfactory receptor protein is embedded into each detection channel; the first electrode group comprises a plurality of electrode pairs, each electrode pair comprises a first working electrode arranged in the independent detection cavity and a first grounding electrode arranged in the public grounding cavity, and the first working electrodes and the first grounding electrodes are used for applying voltage signals and collecting response current signals under the action of the odor molecules to be detected. According to the invention, simultaneous detection of multiple smells can be realized, the detection capability of the bionic olfactory sensor is improved, and the application field of the bionic olfactory sensor is expanded.

Description

Technical Field

[0001] This invention belongs to the field of odor molecule detection technology, specifically relating to a multi-channel sensor, system, and application for detecting odor molecules. Background Technology

[0002] Biomimetic olfactory sensing technology mimics the working principle of the biological olfactory system, utilizing the selective recognition of specific molecules by biological olfactory sensitive materials such as olfactory tissues, olfactory cells, and olfactory receptor proteins to achieve rapid and sensitive detection of odor molecules. Among them, olfactory receptor proteins are diverse, have a broad odor response spectrum, and can perform sensitive detection at the single-molecule level, showing great potential for the development of miniaturized and portable biomimetic olfactory sensors.

[0003] Olfactory receptor proteins from different species possess different structural characteristics. Taking insect olfactory receptor proteins as an example, most insect olfactory receptor proteins are heteromeric multimers with ion channel structures composed of two subunits: the olfactory receptor (OR) and the ORco subunit. The OR subunit has the function of binding to specific small molecules and controlling the opening and closing of ion channels, while the ORco subunit provides support for the heteromeric olfactory receptor protein structure. Some insect olfactory receptor proteins are homomeric ion channels composed of a single subunit, such as the MhOR5 olfactory receptor protein from the rock cricket. These insect olfactory receptor proteins function as both receptors and ion channels. When odor molecules specifically interact with these olfactory receptor proteins, a conformational change occurs, opening the ion channel and promoting the transmembrane flow of related ions, thus converting the chemical information of the odor into an electrical signal. Currently, when constructing biomimetic olfactory sensors using ion channel-type olfactory receptor proteins as sensitive materials, it is necessary to embed the ion channel-type olfactory receptor proteins into a lipid bilayer biomembrane to form odor-gated ion channels on the biomembrane. A multi-channel picoampere-level current detection system is then used to detect and analyze the current response signal under odor stimulation.

[0004] Currently, existing biomimetic olfactory sensors constructed using olfactory receptor proteins as sensitive materials can only achieve single-channel combinations of olfactory receptor proteins and analytes. They can only detect one odor at a time, with limited ability to detect and distinguish multiple odors, and a narrow detection range, failing to simulate the broad-spectrum olfactory capabilities of a biological nose. Since the types of odor molecules detected in the environment are diverse, multiple operations are required when detecting multiple odors. Therefore, for the detection of multiple channels of olfactory receptor proteins and multiple analytes, this type of biomimetic olfactory sensor exhibits low efficiency and repetitive operations, severely limiting its widespread adoption and application. Summary of the Invention

[0005] In view of this, the present invention provides a sensor, system and application for multi-channel detection of odor molecules, which solves the technical problems of the prior art and realizes the simultaneous detection of multiple odors.

[0006] The technical solution of this invention is: A multi-channel sensor for detecting odor molecules, comprising: The detection chamber includes a block with multiple grooves on one end face. The multiple grooves serve as a common grounding cavity and multiple independent detection cavities, respectively. The common grounding cavity and multiple independent detection cavities are used to contain a detection buffer carrying odor molecules to be detected. A detection channel is provided between each independent detection cavity and the common grounding cavity. The detection channel is used to carry a lipid bilayer embedded with olfactory receptor proteins. After the olfactory receptor proteins recognize and bind to the odor molecules to be detected, they adjust the detection channel to switch from a closed state to an open state. The first electrode group includes multiple electrode pairs. Each electrode pair includes a first working electrode disposed in an independent detection cavity and a first grounding electrode disposed in a common grounding cavity. The first working electrode and the first grounding electrode are used to apply a voltage signal and acquire the response current signal under the action of the odor molecule to be detected.

[0007] Furthermore, the common grounding cavity is located at the center of the block, and the multiple independent detection cavities are distributed around the common grounding cavity.

[0008] Furthermore, the detection channel includes a first connecting hole formed on the side wall adjacent to the independent detection cavity and the common grounding cavity, and a Teflon microporous film adhered to the first connecting hole. The Teflon microporous film has a second connecting hole, which is used to form a lipid bilayer and embed olfactory receptor proteins.

[0009] Furthermore, a pre-set electrode base is provided on the lower side of the other end face of the block. The pre-set electrode base includes a base plate and a limiting groove formed on the end face of the base plate for sealing and fixing the position of the block. The limiting groove matches the shape of the block, and the first working electrode and the first grounding electrode are disposed in the limiting groove.

[0010] Furthermore, a sample injection top cover is provided on the upper side of the end face of the block, and the sample injection top cover is sealed to the block for introducing the detection buffer into the common grounding cavity and multiple independent detection cavities respectively.

[0011] Furthermore, the sample injection top cover includes a top plate, on which a first injection hole, multiple second injection holes, and vent holes are provided. The position of the first injection hole corresponds to the common grounding cavity and is used to inject detection buffer into the common grounding cavity. The multiple second injection holes correspond one-to-one with multiple independent detection cavities and are used to inject detection buffer into the independent detection cavities. The multiple vent holes are located on the sides of the multiple second injection holes and are used to balance the internal and external air pressure of the independent detection cavities.

[0012] Furthermore, it also includes multiple cylinders, each corresponding to a multiple second injection hole and coaxially arranged. One end of each cylinder is fixedly connected to the top plate, and the interior of each cylinder forms an independent detection chamber with an internal liquid level lifting channel.

[0013] An odor detection system includes a multi-channel sensor for detecting odor molecules as described above, and also includes a multi-channel current detection unit and a signal processing and analysis unit, wherein the multi-channel current detection unit is electrically connected to the sensor and the signal processing and analysis unit respectively.

[0014] The odor detection system described above is used in customs quarantine and disease screening.

[0015] Compared with the prior art, the present invention provides a multi-channel sensor, system and application for detecting odor molecules. The sensor's detection cavity has a common grounding cavity for accommodating a detection buffer carrying the odor molecule to be detected and multiple independent detection cavities. Each independent detection cavity is provided with a detection channel between the common grounding cavity and the detection channel. The detection channel is used to carry a lipid bilayer embedded with olfactory receptor proteins. After the olfactory receptor proteins recognize and bind to the odor molecule to be detected, they adjust the detection channel to switch from a closed state to an open state, thus forming multiple detection channels. Each detection channel can be used to detect one type of odor molecule. After applying an electrical signal to the first working electrode in the independent detection cavity and the first grounding electrode in the common grounding cavity, the response current signal under the action of the odor molecule to be detected is collected, thereby realizing the detection of the odor molecule to be detected and revealing the type of odor molecule to be detected. This invention utilizes ion channel-type olfactory receptor proteins as sensitive materials to construct a multi-channel biomimetic olfactory sensor, realizing multi-channel parallel odor sensing based on multiple identical / different olfactory receptor proteins. It fully utilizes the combined encoding characteristics of olfactory receptor proteins to achieve simultaneous detection and differentiation of multiple odors, effectively improving the detection capability of the biomimetic olfactory sensor, expanding the application field of biomimetic olfactory sensors, and is highly practical and worthy of promotion. Attached Figure Description

[0016] Figure 1 This is a schematic diagram illustrating the detection principle of a single detection channel of the present invention.

[0017] Figure 2 This is a front view of the overall structure of the present invention.

[0018] Figure 3 This is an exploded view of the overall structure of the present invention.

[0019] Figure 4 This is a top view of the overall structure of the present invention.

[0020] Figure 5 This is a three-dimensional structural diagram of the detection cavity of the present invention.

[0021] Figure 6 This is a top view of the detection cavity of the present invention.

[0022] Figure 7 This is a cross-sectional view of the detection cavity of the present invention. Figure 8 This is a front view of the pre-positioned electrode base of the present invention.

[0023] Figure 9 This is a top view of the pre-positioned electrode base of the present invention.

[0024] Figure 10 This is a three-dimensional structural diagram of the pre-placed electrode base of the present invention.

[0025] Figure 11 This is a schematic diagram of the electrode connections of the pre-set electrode base of the present invention.

[0026] Figure 12 This is a front view of the sample injection top cover of the present invention.

[0027] Figure 13 A top view of the sample injection cap of the present invention.

[0028] Figure 14 This is a three-dimensional structural diagram of the sample injection top cover of the present invention.

[0029] Figure 15 The MhOR5 protein channel of this invention responds to different concentrations of (S)-cis-verbenol.

[0030] Figure 16 This is a schematic diagram illustrating the current response signal characteristic analysis of the present invention.

[0031] Figure 17 The classification results for the three target odors of this invention are shown. Detailed Implementation

[0032] This invention provides a multi-channel sensor, system, and application for detecting odor molecules to solve the aforementioned problems. In order to enable those skilled in the art to better understand and implement the technical solution of this invention, the technical solution of this invention will be clearly and thoroughly described below with reference to the accompanying drawings.

[0033] In the description of this invention, it should be understood that the terms "center," "longitudinal," "lateral," "length," "width," "thickness," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," "clockwise," "counterclockwise," "axial," "radial," and "circumferential" indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are used only for the convenience of describing this invention and simplifying the description, and are not intended to indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this invention.

[0034] Furthermore, it should be further explained that in the description of the embodiments of the present invention, unless otherwise stated, " / " means "or". For example, A / B can mean A or B. The "and / or" in the text is merely a description of the relationship between related objects, indicating that there can be three relationships. For example, A and / or B can mean: A exists alone, A and B exist simultaneously, and B exists alone. In addition, in the description of the embodiments of the present invention, "multiple" means two or more.

[0035] The terms "first," "second," "third," and "fourth" are used for descriptive purposes only and should not be construed as implying or suggesting relative importance or implicitly indicating the number of indicated technical features. Thus, a feature defined as "first," "second," "third," or "fourth" may explicitly or implicitly include one or more of that feature. In the description of this invention, unless otherwise stated, "multiple" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0036] In this invention, unless otherwise explicitly specified and limited, the terms "installation," "connection," "linking," and "fixing," etc., should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral part; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; they can refer to the internal communication of two components or the interaction between two components, unless otherwise explicitly limited. Those skilled in the art can understand the specific meaning of the above terms in this invention according to the specific circumstances.

[0037] In this invention, unless otherwise explicitly specified and limited, "above" or "below" the second feature can mean that the first feature is in direct contact with the second feature, or that the first feature is in indirect contact with the second feature through an intermediate medium. Furthermore, "above," "over," and "on top" of the second feature can mean that the first feature is directly above or diagonally above the second feature, or simply that the first feature is at a higher horizontal level than the second feature. "Below," "below," and "under" the second feature can mean that the first feature is directly below or diagonally below the second feature, or simply that the first feature is at a lower horizontal level than the second feature.

[0038] It should be noted that when an element is referred to as being "fixed to" or "set on" another element, it can be directly on the other element or there may be an intervening element. When an element is considered to be "connected to" another element, it can be directly connected to the other element or there may be an intervening element. The terms "vertical," "horizontal," "upper," "lower," "left," "right," and similar expressions used in this invention are for illustrative purposes only and do not represent the only possible implementation.

[0039] Example 1 An odor detection system includes a multi-channel sensor for detecting odor molecules, a multi-channel current detection unit, and a signal processing and analysis unit, wherein the multi-channel current detection unit is electrically connected to the sensor and the signal processing and analysis unit, respectively.

[0040] The multi-channel sensor for detecting odor molecules is essentially a multi-channel detection chamber with multiple parallel detection channels. Each detection channel consists of a lipid bilayer, olfactory receptor proteins, a detection buffer, and an electrode pair. The lipid bilayer provides a lipid environment for the embedding of olfactory receptor proteins and isolates the detection buffer on both sides. The electrode pair is used to detect the current signal flowing through the lipid bilayer.

[0041] A schematic diagram of the core detection principle within a single detection channel is shown below. Figure 1 As shown, olfactory receptor proteins are embedded in a lipid bilayer. When no odor ligand molecules are present, the detection channel is closed, charged ions cannot flow through the lipid bilayer, and the electrode pair cannot detect a current signal. When odor ligand molecules are present, they act on the olfactory receptor proteins, opening the detection channel. Charged particles flow across the lipid bilayer under the influence of an external electric field, forming a transmembrane current. The electrode pair can detect this current signal. By analyzing the characteristics of the current signal, the detection and differentiation of odor molecules can be achieved.

[0042] The overall structure of the multi-channel sensor for detecting odor molecules is as follows: Figure 2 As shown, Figure 3The diagram shown is an exploded view of the overall structure. From top to bottom, the structure includes a sample injection top cover 3, a detection chamber 1, and a pre-positioned electrode base 2. The detection chamber 1 is designed to facilitate the simultaneous detection of multiple odor molecules using various olfactory receptor proteins, and each channel can achieve independent detection.

[0043] Among them, such as Figure 4 , Figure 5 , Figure 6 and Figure 7 As shown, the detection chamber 1 is made of PMMA or polytetrafluoroethylene through machining. It includes a block 103 and nine grooves formed on one end face of the block 103. The nine grooves form a common grounding cavity 101 and eight independent detection cavities 102. The common grounding cavity 101 and the eight independent detection cavities 102 are used to contain the detection buffer carrying the odor molecules to be detected.

[0044] The bottom of each of the eight independent detection chambers 102 is provided with a working electrode hole 104, and the bottom of the common grounding chamber 101 is provided with a grounding electrode hole 105. The working electrode hole 104 and the grounding electrode hole 105 are used to dock with the pre-set electrode base 2.

[0045] For ease of design and manufacturing, the block 103 can be a cylindrical structure, with the eight independent detection chambers 102 and the common grounding chamber 101 designed as circular grooves. The common grounding chamber 101 is located in the middle of the block 103 and is coaxial with the block 103. The eight independent detection chambers 102 have the same structure and are evenly distributed around the common grounding chamber 101 in a circular array.

[0046] In another embodiment, the eight independent detection cavities 102 and the common grounding cavity 101 are designed as polygonal grooves. The common grounding cavity 101 is located in the middle of the block 103. The eight independent detection cavities 102 have the same structure and are evenly distributed around the common grounding cavity 101 in a circular array.

[0047] Eight independent detection chambers 102 are each connected to a common grounding cavity 101 by a detection channel. Each detection channel includes a first connecting hole on the sidewall between the independent detection chamber 102 and the common grounding cavity 101. A Teflon microporous membrane is attached to the first connecting hole. The Teflon microporous membrane has a second connecting hole with a diameter of approximately 100μm-150μm. The second connecting hole is used to form a lipid bilayer and embed olfactory receptor proteins, so that each independent detection chamber 102 and the common grounding cavity 101 are connected only through the second connecting hole, thus forming eight independent detection channels. The detection channels are used to carry the lipid bilayer with embedded olfactory receptor proteins. The olfactory receptor proteins are used to identify the odor molecules to be detected and bind to them, thereby adjusting the detection channels to switch from a closed state to an open state.

[0048] Specifically, the Teflon microporous film is about 20 micrometers thick, with one and only one second connecting hole in the middle with a diameter of about 100μm-150μm.

[0049] In addition, in order to facilitate fixing with the pre-set electrode base 2, multiple lug structures 106 can be provided around the block 103, and through holes for inserting connectors are provided on the lug structures 106.

[0050] The detection chamber 1 is equipped with a first electrode group, which includes multiple electrode pairs. Each electrode pair includes a first working electrode 201 disposed in an independent detection chamber 102 and a first grounding electrode 202 disposed in a common grounding chamber 101. The first working electrode 201 and the first grounding electrode 202 are used to load an electrical signal onto the detection buffer and collect the response current signal under the action of the odor molecule to be detected.

[0051] A pre-set electrode base 2 is provided on the lower side of the block 103, such as Figure 8 , Figure 9 and Figure 10 As shown, the pre-set electrode base 2 includes a base plate 205 and an electrode lead plate 206. The center of the base plate 205 is a cylindrical cavity 204 with 16 limiting slot openings inside. The positions of the limiting slot openings are coaxially arranged with the working electrode hole 104 and the grounding electrode hole 105, respectively, to ensure that the first working electrode 201 extends into the independent detection cavity 102 and the first grounding electrode 202 extends into the common grounding cavity 101.

[0052] The base plate 205 is provided with four limiting connection holes 203. The four limiting connection holes 203 are located at the four corners of the base plate 205. The positions of the limiting connection holes 203 match the through holes on the lug structure 106. They are used to insert connectors to reliably connect the detection cavity 1 to the pre-positioned electrode base 2, preventing the cavity from loosening and liquid leakage.

[0053] Specifically, bolts and nuts are preferred as connectors.

[0054] Specifically, the wiring of electrode lead plate 206 is as follows: Figure 11 As shown, the first working electrode 201 is vertically connected to the working electrode contact point 208, and the first ground electrode 202 is vertically connected to the ground electrode contact point 207. All working electrodes and ground electrodes are connected to the working electrode external interface 210 and the ground electrode external interface 209 at the edge, thereby ensuring that all the first working electrodes 201 and the first ground electrode 202 are connected to the multi-channel current detection unit, which facilitates the acquisition of multi-channel current data.

[0055] The common grounding cavity 101 provides grounding detection terminals for eight independent detection channels, which are paired with the first working electrode 201 in each of the eight independent detection cavities 102 to form an independent detection channel. The working electrode hole 104 and the grounding electrode hole 105 are used to dock with the preset electrode base 2, and at the same time reliably fix the detection cavity 1 to the preset electrode base 2 to prevent the detection cavity 1 from loosening and the bottom liquid from leaking.

[0056] Specifically, the first working electrode 201 and the first grounding electrode 202 are both silver / silver chloride electrodes. After the silver / silver chloride electrodes are fixed to the electrode lead plate 206 by soldering, the surface of the electrode lead plate 206 is encapsulated with epoxy resin with a thickness of 0.5 mm.

[0057] The preparation of a silver / silver chloride electrode includes the following steps: Take a silver wire of appropriate length, soak it in dilute nitric acid and acetone for 5 minutes respectively to remove the surface oxide layer and oil stains, then polish the surface with 100-grit sandpaper, and soak it in 10% sodium hypochlorite solution for 12 hours to obtain a silver / silver chloride electrode.

[0058] To ensure a tight seal, a sealing insulating gasket is provided on the surface of the electrode lead plate 206 to isolate the electrode lead plate 206 from the independent detection chamber 102, preventing the detection buffer from leaking into the electrode lead plate 206 and interfering with data acquisition during the detection process.

[0059] Among them, a sample injection top cover 3 is provided on the upper side of the end face of the block 103 with multiple grooves. The sample injection top cover 3 is sealed to the block 103 and is used to introduce the detection buffer into the common grounding cavity 101 and multiple independent detection cavities 102 respectively.

[0060] Specifically, the sample injection cap 3 is made of PMMA or polytetrafluoroethylene through machining.

[0061] like Figure 12 , Figure 13 and Figure 14 As shown, the sample injection top cover 3 includes a top plate 301 and a first injection hole 303, a plurality of second injection holes 302 and a vent hole 304 disposed on the top plate 301. The position of the first injection hole 303 corresponds to the common grounding cavity 101 and is used to inject the sample into the common grounding cavity 101. The plurality of second injection holes 302 correspond one-to-one with the plurality of independent detection cavities 102 and are used to inject the sample into the independent detection cavities 102.

[0062] The sample injection top cover 3 also includes multiple cylinders 305, which correspond one-to-one with multiple second injection holes 302 and are coaxially arranged. One end of the cylinder 305 is fixedly connected to the top plate 301. The cylinder 305 forms an internal liquid level lifting channel for an independent detection chamber 102. The internal liquid level lifting channel is used to separate the injection channel from the space inside the detection chamber, and can complete the liquid level lifting operation in each detection chamber with the help of a pipette tip.

[0063] Each second injection port has a vent 304 on its side for balancing the air pressure inside and outside the independent detection chamber 102. The vent is used to balance the air pressure inside and outside the detection chamber, ensuring that the liquid level inside and outside the liquid level lifting channel in the lower chamber of the second injection port is consistent.

[0064] Before odor detection, olfactory receptor proteins need to be added to multiple independent detection chambers 102. The olfactory receptor proteins can be prepared using an E. coli cell-free protein expression system. By adding the gene of the target olfactory receptor protein to the cell-free expression system, the automatic expression of the target olfactory receptor protein can be achieved.

[0065] The formation of the lipid bilayer and the embedding of olfactory receptor proteins are accomplished in the second connecting pores of the Teflon microporous membrane.

[0066] The basic principle and process of lipid bilayer formation are as follows: Each independent detection chamber 102 is separated from the common grounding chamber 101 by a Teflon microporous membrane on the first connecting hole, forming a detection channel. Using RTV3140 adhesive, a Teflon microporous membrane with a second connecting hole is laid flat on the first connecting hole, with the membrane positioned closer to the common grounding chamber 101 to support lipid film formation. This membrane-laying step is only required for the first use; after laying, the Teflon microporous membrane can be reused repeatedly without frequent replacement. After the detection channel membrane is laid, a certain volume of detection buffer solution is injected into the independent detection chamber 102 and the common grounding chamber 101 on both sides of the second connecting hole, ensuring that the liquid level of the detection buffer solution remains below the height of the second connecting hole. Then, an appropriate amount of lipid solution with a concentration of 2 mg / mL-10 mg / mL is added dropwise to the independent detection chamber 102 and the common grounding chamber 101 respectively. The lipid molecules will spread on the horizontal surface of the detection buffer solution to form a lipid monolayer.

[0067] Detection buffer is injected into both the independent detection chamber 102 and the common grounding chamber 101, raising the liquid level above the location of the second connecting hole. At this point, the lipid monolayers on both sides of the second connecting hole will spontaneously form a lipid bilayer (hereinafter referred to as a lipid membrane) at the second connecting hole under the influence of surface tension and hydrophobic interaction. The formation and quality of the lipid membrane can be evaluated by the current in the detection channel. After the lipid membrane is formed, an olfactory receptor protein is added to an independent detection chamber 102. Because the olfactory receptor protein has hydrophobic domains, it can spontaneously embed into the lipid membrane, thus ensuring that a single ion channel-type olfactory receptor protein is embedded in the lipid membrane.

[0068] The principle for characterizing successful lipid film formation is as follows: Successful lipid film formation can be considered as an RC parallel circuit, with the resistance approximately infinite. After inputting a triangular wave signal, a square wave signal can be detected as the output response. The amplitude of the square wave signal reflects the quality of the formed lipid film. When the lipid film is successfully formed, an input triangular wave signal with a frequency of 10 Hz and a peak-to-peak value of 1 volt will detect a square wave current amplitude of approximately 100 picoamperes.

[0069] Before use, a pretreatment solution is required to perform a hydrophobic treatment on the second connecting pore area of ​​the Teflon microporous membrane.

[0070] The specific procedure for surface hydrophobic treatment is as follows: apply a pretreatment solution of about 5 microliters to the Teflon microporous membrane that is attached during the film application step and wait for 5 minutes for the pentane to evaporate naturally.

[0071] Specifically, the pretreatment solution is a mixed solution of pentane and hexadecane, wherein the mass percentage of hexadecane in the mixture is 1%–5%. The detection buffer is an aqueous solution containing NaCl (96 mmol / L), MgCl2 (5 mmol / L), KCl (2 mmol / L), CaCl2 (0.8 mmol / L), and 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (5 mmol / L), with a pH of 7–8. The solute in the lipid solution is DPhPC, with a mass concentration of 1–10 mg / mL, and the solvent is pentane.

[0072] DPhPC is a synthetic phosphatidylcholine derivative, its full name being 1,2-Diphytanoyl-sn-glycero-3-phosphocholine. Available lipids include, but are not limited to, DOPC, DPhPC, DMPC, and lecithin; different lipids can be used in combination.

[0073] By applying a specific clamping voltage to the lipid membrane through the first working electrode 201 and the first ground electrode 202, the gated current signal of the olfactory receptor protein under the stimulation of odor molecules can be detected.

[0074] The multi-channel current detection unit is a multi-channel picoampere-level current detection system, directly connected to the electrode lead plate 206, with multiple detection channels detecting in parallel. For a single detection channel, when a certain clamping voltage, such as -60 millivolts, is applied between the first working electrode 201 and the first ground electrode 202, the corresponding current response signal is detected, thereby realizing the recording and analysis of the test current.

[0075] The recorded multi-channel current response signals directly reflect the opening and closing states of ion channels triggered by odor molecule stimulation. In the closed state, the current is at a baseline of 0. When the ion channel opens, charged ions move directionally under the drive of a clamping voltage, generating a corresponding response current signal. Signal processing and analysis primarily involve first addressing the multi-dimensional response parameters corresponding to these opening / closing events, including current response amplitude and variance, opening duration, closing duration, and response frequency over a certain time period. Then, the correlation between these response parameters and the type and concentration of odor stimuli is analyzed.

[0076] The multi-channel current detection unit uses a commercial or customized multi-channel picoampere-level current detection system to achieve parallel detection of picoampere-level currents in multiple channels. Each channel corresponds to a pair of detection electrodes, which are connected to the multi-channel current detection unit through an interface, thereby realizing the detection of the current response signal of each channel. The multi-channel current signals are uploaded to the signal processing and analysis unit for subsequent data analysis and processing.

[0077] Specifically, the signal processing and analysis unit is implemented using computer software.

[0078] The method of using an odor detection system is as follows: Step 1: Install the Teflon microporous membrane with the second connecting hole onto the first connecting hole of the detection chamber 1. Fix the detection chamber 1 onto the pre-set electrode base 2 using a connector. Insert the corresponding first working electrode 201 and first grounding electrode 202 into the pre-reserved working electrode hole 104 and grounding electrode hole 105 of the detection chamber 1, respectively. Tighten the connector until the detection chamber 1 and the pre-set electrode base 2 are in a completely sealed connection state. Connect the electrodes to the multi-channel current detection unit through the pre-set electrode base 2.

[0079] Step 2: Use pretreatment solution to treat the Teflon microporous membrane of each channel separately. Add about 0.5 mL of detection buffer to the common grounding cavity 101 and each independent detection cavity 102 respectively, and then add lipid solution. By raising the liquid level of each independent detection cavity 102, a lipid bilayer is formed at the second connecting pore of the Teflon microporous membrane.

[0080] 0.5 mL of detection buffer solution is injected into the common grounding cavity 101 and each independent detection cavity 102 through the first injection hole 303 and the second injection hole 302 on the sample injection top cover 3, respectively.

[0081] An appropriate amount of lipid solution is added to the common grounding cavity 101 and each independent detection cavity 102 through the first injection hole 303 and the second injection hole 302, respectively, so that the lipids are horizontally spread on the surface of the detection buffer solution.

[0082] Approximately 0.7 mL of detection buffer solution is slowly added to the common grounding cavity 101 and each independent detection cavity 102 through the first injection hole 303 and the second injection hole 302, respectively, so as to slowly raise the level of the detection buffer solution in each cavity.

[0083] Step 3: Apply triangular wave voltage signals to each channel using a function generator, and evaluate the formation quality of the lipid bilayer by the response current signals.

[0084] When the parameters of the triangular wave voltage are 10 Hz and the peak-to-peak value is 1 volt, the output response current signal is a square wave signal of approximately ±100 picoamperes, which can be used to determine the successful formation of the lipid bilayer.

[0085] Step 4: Add olfactory receptor protein solution to each independent detection chamber 102, and wait for it to spontaneously embed into the lipid bilayer to form ion channels that can respond to odors, which are used to detect the odor molecules to be detected.

[0086] Specifically, olfactory receptor protein solution is added to each independent detection chamber 102 through the second injection hole 302. The concentration of olfactory receptor protein is 100 micrograms / mL and the volume is 1-10 microliters. The same olfactory receptor protein solution can be added to each independent detection chamber 102, or different olfactory receptor protein solutions can be added according to the settings to form a multi-channel olfactory receptor protein array.

[0087] It should be noted that the olfactory receptor proteins used in this example are insect olfactory receptor proteins MhOR5, ItypOR23 / ORCO, and ItypOR25 / ORCO, where MhOR5 is a homotetramer, and ItypOR23 / ORCO and ItypOR25 / ORCO form heterotetramers respectively.

[0088] Step 5: A test solution containing the odor molecules to be detected is added to each independent detection chamber 102. After the olfactory receptor protein binds to the specific odor molecules, the protein conformation changes, which open the ion channel and generate an ion response current. The multi-channel current detection unit applies a bias voltage to each detection channel through the first working electrode 201 and the first ground electrode 202 to detect the response current signal under the action of odor molecules.

[0089] Specifically, the bias voltage can be adjusted within the range of ±100 millivolts. If the voltage is too low, the corresponding response current will be too small, and if the voltage is too high, it may cause the lipid bilayer to break down. In this example, -60 millivolts is used.

[0090] The odor molecules to be detected include α-pinene, 2-methyl-3-buten-2-ol, S-cis-verbenol, etc.

[0091] Step 6: Analyze and process the current response signal, extract the features of the response signal, and establish an odor-response dataset for the detection and analysis of target odors.

[0092] Specifically, the method for analyzing and processing the current response signal is as follows: In the absence of odor molecule stimulation, the electrical signal is at a baseline level of 0. When odor molecule stimulation is applied, it causes the olfactory receptor protein channels to open, thereby producing a signal like... Figure 15 The current response signal shown corresponds to the opening and closing of the ion channel. Its characteristics include amplitude, duration, current standard deviation, and event frequency. Figure 16 As shown.

[0093] The characteristics of the response signal are jointly determined by factors such as the type and concentration of the odor molecules being tested, and the nanopore properties of the olfactory receptor proteins. Current response signal features from multiple channels are extracted to establish an odor-response dataset. This dataset is then trained using a deep learning algorithm to detect the odor molecules being tested, revealing the types of odor molecules being detected, such as... Figure 17 As shown.

[0094] Example 2 As a parallel optimization scheme, in order to reduce workload and optimize structure, the first electrode group is replaced by the second electrode group. The other structures are the same as in Example 1. The second electrode group includes multiple second working electrodes arranged one-to-one in the independent detection cavity 102 and a second grounding electrode arranged in the common grounding cavity 101. The second working electrode and the second grounding electrode are planar silver / silver chloride electrodes. The second working electrode and the second grounding electrode are used to input electrical signals into the detection buffer and collect the response current signal under the action of the odor molecules to be detected.

[0095] In this configuration, the limiting groove matches the shape of the block 103. One end of the second working electrode and the second grounding electrode are exposed to the independent detection cavity 102 and the common grounding cavity 101, respectively, and the other end is electrically connected to the electrode lead plate 206 to concentrate the signal of the second electrode group for easy multi-channel data acquisition.

[0096] The multi-channel sensor for detecting odor molecules based on olfactory receptor proteins provided by this invention has strong scalability and can be applied to various ion channel type olfactory receptor proteins and the odor molecules to be detected. It can be used in odor detection-related fields such as customs quarantine and disease screening.

[0097] This invention provides a multi-channel sensor for detecting odor molecules. Its detection chamber has a common grounding cavity for accommodating a detection buffer solution carrying the odor molecules to be detected, and multiple independent detection cavities. Each independent detection cavity is connected to the common grounding cavity by a detection channel. The detection channel carries a lipid bilayer and embeds olfactory receptor proteins, thus forming multiple detection channels. Each detection channel can be used to detect one type of odor molecule. After applying electrical signals to the first working electrode 201 in the independent detection cavity and the first grounding electrode 202 in the common grounding cavity, the response current signal under the action of the odor molecule to be detected is acquired, thereby realizing the detection of the odor molecule and revealing its type. This invention utilizes ion channel-type olfactory receptor proteins as sensitive materials to construct a multi-channel biomimetic olfactory sensor, achieving multi-channel parallel odor sensing based on multiple identical / different types of olfactory receptor proteins. It fully utilizes the combined encoding characteristics of olfactory receptor proteins to achieve simultaneous detection and differentiation of multiple odors, effectively improving the detection capability of the biomimetic olfactory sensor, expanding the application field of biomimetic olfactory sensors, and demonstrating strong practicality, making it worthy of promotion.

[0098] The above-disclosed embodiments are merely preferred embodiments of the present invention. However, the embodiments of the present invention are not limited thereto, and any variations that can be conceived by those skilled in the art should fall within the protection scope of the present invention.

Claims

1. A multi-channel sensor for detecting odor molecules, characterized in that, The utility model relates to a detection device for olfactory receptor protein, comprising: a detection cavity (1) comprising a block (103), a plurality of recesses are arranged on one end face of the block (103), and the plurality of recesses are used as a common ground cavity (101) and a plurality of independent detection cavities (102) respectively, the common ground cavity (101) and the plurality of independent detection cavities (102) are used for accommodating detection buffer solution carrying odor molecules to be detected, a detection channel is arranged between each independent detection cavity (102) and the common ground cavity (101), the detection channel is used for carrying a lipid bilayer embedded with olfactory receptor proteins, the olfactory receptor proteins are used for identifying the odor molecules to be detected and adjusting the detection channel from a closed state to an open state after being combined with the odor molecules to be detected; a first electrode group comprising a plurality of electrode pairs, each electrode pair comprising a first working electrode (201) arranged in the independent detection cavity (102) and a first ground electrode (202) arranged in the common ground cavity (101), the first working electrode (201) and the first ground electrode (202) are used for applying a voltage signal and collecting a response current signal under the action of the odor molecules to be detected.

2. The sensor according to claim 1, wherein The common ground cavity (101) is located at a central position of the block (103), and the plurality of independent detection cavities (102) are uniformly distributed around the common ground cavity (101).

3. The sensor of claim 2, wherein, The detection channel comprises a first communication hole arranged on a side wall adjacent to the independent detection cavity (102) and the common ground cavity (101), and a Teflon microporous film arranged on the first communication hole, the Teflon microporous film has a second communication hole, and the second communication hole is used for forming a lipid bilayer and embedding olfactory receptor proteins.

4. The sensor of claim 1, wherein, The other end face of the block (103) is provided with a preset electrode base (2) on the lower side, the preset electrode base (2) comprises a bottom plate (201) and a limiting groove arranged on the end face of the bottom plate (201) and used for sealingly fixing the position of the block (103), the limiting groove is matched with the shape of the block (103), and the first working electrode and the first ground electrode are arranged in the limiting groove.

5. The sensor of claim 4, wherein, A sample injection top cover (3) is arranged on the upper side of the end face of the block (103), the sample injection top cover (3) is sealingly connected with the block (103) and is used for guiding the detection buffer solution into the common ground cavity (101) and the plurality of independent detection cavities (102) respectively.

6. The sensor of claim 5, wherein, The sample injection top cover (3) comprises a top plate (301), a first liquid injection hole, a plurality of second liquid injection holes and a vent hole are arranged on the top plate (301), the position of the first liquid injection hole corresponds to the common ground cavity (101) and is used for injecting the detection buffer solution into the common ground cavity (101), the plurality of second liquid injection holes correspond to the plurality of independent detection cavities (102) one by one and are used for injecting the detection buffer solution into the independent detection cavities (102), and the plurality of vent holes are arranged on the side edges of the plurality of second liquid injection holes and are used for balancing the internal and external air pressures of the independent detection cavities (102).

7. The sensor of claim 6, wherein, The application further comprises a plurality of barrels (302) which are coaxially arranged with the plurality of second liquid injection holes one by one, one end of the barrel (302) is fixedly connected with the top plate (301), and a cavity inner liquid level lifting channel of the independent detection cavity (102) is formed inside the barrel (302).

8. An odor detection system characterized by, The application further comprises a multi-channel current detection unit and a signal processing and analysis unit, and the multi-channel current detection unit is electrically connected with the sensor and the signal processing and analysis unit respectively.

9. The odor detection system of claim 8, wherein, The application is used in customs quarantine and disease screening.

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