Nanopore sensor based on insect olfactory receptor protein as well as construction method and application of nanopore sensor

Abstract

The invention discloses a nanopore sensor based on insect olfactory receptor protein as well as a construction method and application thereof, and belongs to the field of biosensors. According to the invention, the insect olfactory receptor protein is used as a sensitive element to be integrated into a nanopore measurement system, so that the nanopore sensor is obtained. By measuring a nanopore current signal, the prepared sensor is applied to detection of target odorant molecules. According to the method, the recognition capability of the insect olfactory receptor protein on odorant molecules and the single molecule level detection capability of the nanopores are utilized, expensive large experimental instruments and complex experimental operation steps are not needed, and sensitive, rapid and direct detection on odorant substances can be realized.

Description

Technical Field

[0001] The present invention belongs to the field of biosensors, and in particular relates to a nanopore sensor based on insect olfactory receptor protein and a construction method and application thereof. Background Art

[0002] Insects possess an extremely sensitive sense of smell, enabling them to identify and detect a wide range of odor molecules in complex environments. Olfactory receptor proteins play a crucial role in this process. These proteins are a type of ligand-gated ion channel, primarily found on the surface of olfactory neurons. They bind to odor molecules, generating olfactory perception and capable of detecting single molecules.

[0003] Inspired by the insect olfactory perception system, research on sensors using natural insect-derived biosensors, using methods such as quartz crystal microbalances, surface plasmon resonance, and electrochemical impedance spectroscopy, has been conducted. However, these sensors still fall short of expectations in terms of detection level and sensitivity. Related research primarily uses odorant-binding proteins, while fewer studies have used olfactory receptors as sensitive materials to construct sensors. Furthermore, bionanopore technology, with its advantages of single-molecule detection and direct, rapid detection, has been successfully applied to single-molecule sequencing and biosensing. However, its application in odor detection remains limited.

[0004] Therefore, by utilizing the sensitive recognition ability of insect olfactory receptor proteins for odor molecules and the detection advantage of nanopores at the single-molecule level, insect olfactory receptors are used as biological nanopores to develop a nanopore sensor based on insect olfactory receptor proteins, in order to achieve highly sensitive, rapid and direct detection of odor substances. Summary of the Invention

[0005] The purpose of the present invention is to provide a nanopore sensor based on insect olfactory receptor protein and its construction method and application in response to the deficiencies of the existing technology.

[0006] The objectives of the present invention are achieved through the following technical solutions: In the first aspect, a nanopore sensor based on an insect olfactory receptor protein is provided, wherein the sensor includes two chambers filled with an electrolyte solution, the two chambers are separated by a separator with micropores, and each chamber has an integrated electrode; a phospholipid bilayer is constructed at the micropore, and the insect olfactory receptor protein is embedded in the phospholipid bilayer to serve as the nanopore of the sensor; wherein the insect olfactory receptor protein is used in the form of a homopolymer.

[0007] Furthermore, a phospholipid bilayer is constructed at the micropores, and the insect olfactory receptor protein is embedded in the phospholipid bilayer, which is obtained by the following steps: mixing the insect olfactory receptor protein with phospholipid and incubating it to obtain an insect olfactory receptor protein-phospholipid mixture, and coating the micropores with a bubble method.

[0008] In a second aspect, a method for preparing a nanopore sensor based on an insect olfactory receptor protein is provided, comprising the following steps: (1) Preparation of insect olfactory receptor protein-phospholipid mixture: An insect olfactory receptor protein solution with a concentration of 0.32 mg / ml was mixed with a 10 mg / ml DphPC (diphytylphosphatidylcholine) phospholipid solution in equal volumes and incubated at 4°C for 16 h to obtain an insect olfactory receptor protein-phospholipid mixture. The solute of the insect olfactory receptor protein solution was insect olfactory receptor protein, and the solvent was: 150 mM sodium chloride solution, 20 mM Tris (tris(hydroxymethyl)aminomethane)) buffer, pH 8.0. The concentration represents the final concentration. The solute of the DphPC phospholipid solution was DphPC, and the solvent was n-octane.

[0009] (2) Construction of a nanopore sensor based on insect olfactory receptor protein: The insect olfactory receptor protein-phospholipid mixture obtained after incubation is coated on the micropores of the measuring chip to form a double-layer membrane, thereby obtaining a nanopore sensor based on insect olfactory receptor protein.

[0010] The measurement chip comprises two chambers, each containing an independent integrated Ag / AgCl electrode for collecting time-varying current signals. The chambers contain an electrolyte solution (the concentration is the final concentration in the electrolyte solution): 135 mM NaCl, 5 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 10 mM Hepes, pH 7.3. The micropores of the measurement chip have a diameter of 100 µm, and a double-layer membrane is coated at the micropores with an insect olfactory receptor protein-phospholipid mixture.

[0011] When the insect olfactory receptor protein-phospholipid mixture is coated on the measurement chip, the insect olfactory receptor protein is embedded in the phospholipid bilayer membrane. The bilayer membrane separates the electrolyte solutions in the two chambers of the measurement chip, and the insect olfactory receptor protein provides a channel connecting the two chambers of the measurement chip.

[0012] The insect olfactory receptor protein can be assembled into a homotetrameric odor-gated ion channel; under the action of a target odor ligand, the insect olfactory receptor protein is in an open structural state, and the pore diameter at the opening of the open structural state is about 1 nm; the nanopore is an insect olfactory receptor protein.

[0013] Furthermore, after the insect olfactory receptor protein-phospholipid mixture obtained after incubation is coated on the micropores of the measurement chip, an electrophysiological experiment can be performed by introducing a working concentration of eugenol into the electrolyte solution to determine whether the insect olfactory receptor protein is embedded in the double-layer membrane: if a current change is found, it means that the receptor protein has been embedded in the double-layer membrane.

[0014] In a third aspect, an application of the nanopore sensor based on insect olfactory receptor protein in detecting floral fragrance substances is provided: odor molecules in the air to be tested are sampled and transferred to the electrolyte solution of the nanopore sensor, a voltage is applied between the electrodes, and the current signal is recorded. If a current change is detected relative to the baseline current, it is determined that the air to be tested contains floral fragrance substances.

[0015] More preferably, eugenol is first added to the electrolyte solution of the nanopore sensor and configured to a working concentration, and the current change amplitude is detected as a standard value; then the air to be tested is detected and the current signal is recorded. If the detected current change amplitude is higher than the standard value, it is determined that the air to be tested contains the floral scent substance eugenol.

[0016] The insect olfactory receptor protein is a homotetrameric insect olfactory receptor protein MhOR5, which comes from a ground-dwelling insect called Machilis hrabei.

[0017] In some embodiments of the present invention, the following experiments were performed: (1) Prepare the target test sample solution: dissolve the target molecule to be detected in DMSO to prepare a target molecule stock solution, and use an electrolyte solution to dilute the target molecule stock solution to the working concentration.

[0018] (2) Characterization of phospholipid bilayer membrane formation: The measurement chip is connected to a lipid bilayer recording device, an electrolyte solution is added to the chambers on both sides of the measurement chip, and a phospholipid bilayer membrane is coated on the micropores of the measurement chip using a phospholipid solution or an insect olfactory receptor protein-phospholipid mixture. The lipid bilayer recording device is used for electrophysiological experimental measurements, which detect and record the current signal changing with time. When the phospholipid bilayer membrane is formed, the two chambers of the measurement chip are in an open circuit state, the output current is 0 pA, and the membrane capacitance of the phospholipid bilayer membrane is: 20-50 pF (100 µm pore measurement chip).

[0019] (3) Detecting a solution containing the target odor molecule: Add the target solution to one side of the measurement chip chamber, apply a +50 mV voltage, and record the current signal changes over time before and after the addition of the target molecule at a 5 kHz sampling frequency and a 1 kHz low-pass filter. Analyze the data to identify the target molecule.

[0020] Wherein, the target molecule is eugenol, the concentration of the target molecule stock solution is 150 mM; and the working concentration is 5 µM.

[0021] Among them, the use of a phospholipid solution to coat a phospholipid bilayer membrane at the micropores of the measuring chip is used as a control experiment for using an insect olfactory receptor protein-phospholipid mixture to coat a phospholipid bilayer membrane at the micropores of the measuring chip. The control experiment is used to illustrate that the change in the current signal comes from the interaction between the insect olfactory receptor protein and the target odor molecule.

[0022] The beneficial effects of the present invention are as follows: (1) Olfactory receptors derived from insects can recognize different odor molecules, giving insects highly sensitive olfactory perception. Compared with traditional artificial materials, insect olfactory receptors can detect odor molecules with high selectivity and high sensitivity.

[0023] (2) The present invention creatively uses homotetrameric insect olfactory receptor proteins to construct nanopore sensors, and accordingly develops a rapid and convenient membrane insertion method suitable for homotetramers. Specifically, the present invention utilizes the insect olfactory receptor MhOR5 from Machilis hrabei, which is an odor-gated ion channel assembled in the form of a homotetramer. Compared with other heteromeric insect olfactory receptors, MhOR5 is composed only of odorant receptor (OR) subunits and does not contain odorant receptor co-receptor (Orco) subunits. The selection of MhOR5 for sensor development reduces the complexity of olfactory receptor protein preparation, thereby making sensor construction easier.

[0024] (3) Nanopores can achieve single-molecule detection. Using MhOR5 as a nanopore, we can utilize MhOR5's natural response to odor molecules to detect odors at the single-molecule level. However, protein nanopores commonly used in existing technologies usually require additional protein engineering or chemical modification to achieve specific functional applications.

[0025] (4) The nanopore sensor constructed by the present invention is easy to operate and takes a short time. It does not require any large instruments or complicated data processing procedures and can achieve rapid identification and direct detection of target substances. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the SDS-PAGE (Sodium Dodecyl Sulfate -Polyacrylamide Gel Electrophoresis) result of the insect olfactory receptor protein MhOR5; Figure 2 Schematic diagram of the construction of nanopore sensors based on insect olfactory receptor proteins and their application in odor detection; Figure 2 A in the figure is a flowchart for the construction of nanopore sensors based on insect olfactory receptor proteins. Figure 2 B in the figure is a schematic diagram of the state of insect olfactory receptor proteins when there is no target odor molecule. Figure 2 C in the figure is a schematic diagram showing the change of current over time when there is no target odor molecule. Figure 2 D in the figure is a schematic diagram of the state of insect olfactory receptor proteins when the target odor molecule is present. Figure 2 E in the figure is a schematic diagram showing the change of current over time when the target odor molecules are present; Figure 3 Schematic diagram of a representative continuous current trace at +20 mV when a bilayer membrane is formed on the nanopore measurement chip; Figure 4 Schematic diagram of the continuous current trace of the MhOR5-based nanopore sensor at a voltage of +50 mV in the absence of target odor molecules; Figure 5 Schematic diagram of the continuous current trace of the MhOR5-based nanopore sensor detecting eugenol; Figure 6 for Frequency distribution histogram of ; Figure 7 Schematic diagram of the continuous current trace of electrophysiological detection of eugenol in the absence of MhOR5. DETAILED DESCRIPTION

[0027] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0028] Example 1: Preparation of insect olfactory receptor protein-phospholipid mixture.

[0029] The insect olfactory receptor protein used in this example is MhOR5 (PDB ID: 7LIC), which was synthesized by Dima Biotech (Wuhan). Analysis of the MhOR5 structure revealed that the insect olfactory receptor protein assembles into a homotetrameric ion channel. When bound to a target odorant molecule, the top of MhOR5 transforms from a hydrophobic constriction to a pore with a diameter of 9.2. The ion channel is opened and in an open state, allowing the passage of hydrated ions. Figure 1 shown.

[0030] An insect olfactory receptor protein-phospholipid mixture was prepared for subsequent construction of the sensor bilayer membrane. First, DphPC phospholipid was dissolved in n-octane (purity ≥99%) to prepare a 10 mg / mL DphPC phospholipid solution. To this solution, 5 µL of the DphPC phospholipid solution at 0.32 mg / mL was added. The mixture was thoroughly mixed and incubated overnight at 4°C for 16 hours to obtain the insect olfactory receptor protein-phospholipid mixture. The solvent for the insect olfactory receptor protein solution consisted of 150 mM sodium chloride solution and 20 mM Tris (tris(hydroxymethyl)aminomethane)) buffer, pH 8.0.

[0031] Example 2: Construction of an electrophysiological detection platform.

[0032] The nanopore measurement chip is used for the subsequent reconstitution of insect olfactory receptor proteins on a phospholipid bilayer membrane. First, the nanopore measurement chip is washed with ultrapure water and ethanol, respectively, and then dried under nitrogen. The chip's two measurement chambers are then filled with an electrolyte solution composed of ultrapure water: 135 mM NaCl, 5 mM KCl, 2 mM MgCl₂, 2 mM CaCl₂, and 10 mM Hepes (4-hydroxyethylpiperazineethanesulfonic acid) buffer, pH 7.3. The chip is then connected to an electrophysiological readout (current amplifier) ​​for real-time measurement and recording of ion channel events with ultralow noise. The nanopore measurement chip and electrophysiological readout can be performed using Elements' BLM Chip and the fully integrated handheld portable Nanopore Reader 100 kHz.

[0033] Example 3: Formation of phospholipid bilayer membrane and insertion of insect olfactory receptor protein into the membrane.

[0034] By assembling a phospholipid bilayer on a nanopore measurement chip and embedding insect olfactory receptor proteins, a nanopore sensor based on insect olfactory receptor proteins was constructed to detect target odor molecules by measuring changes in channel current ( Figure 2). Use the phospholipid solution obtained in Example 1 or the insect olfactory receptor protein-phospholipid mixture after incubation to form a double-layer membrane on the micropores of the measuring chip after treatment in Example 2, and check the film-forming state. Among them, the coating preferably adopts the bubble method, specifically the lipid-covered bubble method, and the specific steps are as follows: immerse the tip of the pipette tip in the phospholipid solution obtained in Example 1 or the insect olfactory receptor protein-phospholipid mixture after incubation, dip the liquid, and then use the pipette tip to generate air bubbles at a position close to the micropores of the measuring chip. The air bubbles are in contact with the micropores of the measuring chip, so that the lipids or lipid mixture covering the surface of the air bubbles are coated on the micropores to form a double-layer membrane. When the phospholipid bilayer membrane is formed, the two chambers of the measuring chip are in an open circuit state, and the output current is 0 pA ( Figure 3 The typical membrane capacitance of a phospholipid bilayer membrane is 20-50 pF (measured on a 100 µm pore chip). For control experiments, a bilayer membrane was formed using a phospholipid solution. When a bilayer membrane was formed using an insect olfactory receptor protein-phospholipid mixture, the insect olfactory receptor protein was embedded in the phospholipid bilayer membrane.

[0035] Example 4: Electrophysiological characterization of the nanopore sensor based on insect olfactory receptor protein.

[0036] The sensor prepared in Example 3 was characterized by electrophysiological measurement. A voltage of +50 mV was set, and the current signal was recorded over time in an electrolyte solution environment before the target molecule was added under a sampling frequency of 5 kHz and a low-pass filter of 1 kHz. Figure 4 As shown, in the absence of target molecules, the output current remains at 0 pA and no channel opening events of the MhOR5 insect olfactory receptor protein are observed.

[0037] Example 5: Detection of floral scent odor molecules using a nanopore sensor based on insect olfactory receptor proteins.

[0038] The eugenol to be tested was dissolved in DMSO to prepare a 150 mM eugenol stock solution. The eugenol stock solution was diluted 100-fold with the electrolyte solution prepared in Example 2 for later use. The diluted eugenol solution was then added to the electrolyte solution in the chamber of the measurement chip characterized in Example 4 to achieve a working concentration of 5 µM eugenol. Data were recorded using the same electrophysiological measurement conditions as in Example 4. Figure 5 As shown, for the nanopore sensor based on insect olfactory receptor protein, when the target odor molecule eugenol is added, the channel opening event generated by the binding of MhOR5 insect olfactory receptor protein to eugenol can be observed, wherein ( ) is the ion channel open current ( ) and ion channel closing current ( ) represents the event amplitude of the ion channel opening event, such as Figure 6 As shown in (the histogram is Gaussian fitted and the fitting line is marked with a solid line), the ion channel current corresponding to this event increases; Figure 7 As shown, in the absence of insect olfactory receptor proteins (using only the phospholipid solution to construct the bilayer membrane for subsequent electrophysiological measurements as described in Example 3), no ion channel activity was observed after the addition of eugenol, and the output current remained at 0 pA. These results indicate that the change in the current signal originates from the interaction between the insect olfactory receptor protein and the target odor molecule, verifying that the nanopore sensor constructed based on the insect olfactory receptor protein can be applied to the detection of target odor molecules.

[0039] In summary, the present invention integrates insect olfactory receptor proteins as sensitive elements into a nanopore measurement system by embedding them into a phospholipid bilayer membrane. Nanopore electrophysiological measurement experiments are then used to measure the current signals of the insect olfactory receptor proteins in response to odor molecules. Based on the changes in the detected current signals, direct and rapid detection of target odor molecules is achieved.

Claims

1. A nanopore sensor based on insect olfactory receptor protein, characterized in that: The sensor includes two chambers filled with electrolyte solution, separated by a separator with micropores, and each chamber integrates an electrode; a phospholipid bilayer is constructed at the micropores, and an insect olfactory receptor protein is embedded in the phospholipid bilayer to serve as the nanopore of the sensor; wherein the insect olfactory receptor protein is in the form of a homomer.

2. The nanopore sensor according to claim 1, wherein A phospholipid bilayer is constructed at the micropores, and the insect olfactory receptor protein is embedded in the phospholipid bilayer. The method is obtained by mixing the insect olfactory receptor protein with phospholipids and incubating them to obtain a mixture of the insect olfactory receptor protein and phospholipids, and then coating the mixture on the micropores using a bubble method.

3. The nanopore sensor according to claim 2, characterized in that The concentration of the insect olfactory receptor protein solution was 0.32 mg / ml, and the concentration of the phospholipid solution was 10 mg / ml. The two were mixed in equal volumes and incubated at 4°C for 16 hours to obtain a mixture of the insect olfactory receptor protein and phospholipid.

4. The nanopore sensor according to claim 2, characterized in that The phospholipid is diphytylphosphatidylcholine.

5. The nanopore sensor according to claim 1, wherein The micropore diameter is 100 μm.

6. The nanopore sensor according to claim 1, characterized in that The electrolyte solution is an alkali metal halide aqueous solution.

7. Use of the nanopore sensor according to any one of claims 1 to 6 in detecting floral aroma substances, characterized in that: The insect olfactory receptor protein adopts the homotetrameric insect olfactory receptor protein MhOR5.

8. The use according to claim 7, characterized in that The application specifically includes: sampling odor molecules in the air to be tested, transferring them to the electrolyte solution of the nanopore sensor, applying voltage between electrodes, recording the current signal, and if a current change is detected relative to the baseline current, it is determined that the air to be tested contains floral fragrance substances.

9. The use according to claim 7, characterized in that The floral aroma substance is eugenol.

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