Nanoporous sensors based on insect olfactory receptor proteins, their construction methods and applications

Abstract

This invention discloses a nanopore sensor based on insect olfactory receptor proteins, its construction method, and its applications, belonging to the field of biosensors. This invention obtains the nanopore sensor by integrating insect olfactory receptor proteins as sensitive elements into a nanopore measurement system. By measuring the nanopore current signal, the prepared sensor is applied to the detection of target odor molecules. This invention utilizes the recognition ability of insect olfactory receptor proteins for odor molecules and the single-molecule level detection capability of nanopores, achieving sensitive, rapid, and direct detection of odor substances without the need for expensive, large-scale experimental equipment and complex experimental procedures.

Description

Technical Field

[0001] This invention belongs to the field of biosensors, specifically relating to nanopore sensors based on insect olfactory receptor proteins, their construction methods, and applications. Background Technology

[0002] Insects possess an extremely sensitive sense of smell, enabling them to identify and detect a variety of different odor molecules in complex environments. Insect olfactory receptor proteins play a crucial role in this process. These proteins belong to a type of ligand-gated ion channel and are primarily located on the surface of olfactory nerve cells. They bind to odor molecules, generating olfactory perception and achieving detection at the single-molecule level.

[0003] Inspired by the olfactory perception system of insects, current research on sensors utilizing natural biological sensitive materials derived from insects, based on methods such as quartz crystal microbalances, surface plasmon resonance, and electrochemical impedance spectroscopy, has been conducted. However, their detection levels and sensitivity still fall short of requirements. Related research mainly uses odor-binding proteins, with fewer studies utilizing olfactory receptors as sensitive materials to construct sensors. In addition, while bio-nanopore technology, with its advantages of single-molecule level and direct, rapid detection, has been successfully applied to single-molecule sequencing and biosensing, its application in odor detection remains a gap.

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

[0005] The purpose of this invention is to address the shortcomings of existing technologies by providing a nanopore sensor based on insect olfactory receptor proteins, its construction method, and its application.

[0006] The objective of this invention is achieved through the following technical solution: In a first aspect, a nanopore sensor based on insect olfactory receptor protein is provided. The sensor includes two chambers filled with an electrolyte solution, the two chambers being separated by a separator with micropores, and each chamber integrating an electrode. A phospholipid bilayer is constructed at the micropore, and the insect olfactory receptor protein is embedded in the phospholipid bilayer as the nanopore of the sensor. The insect olfactory receptor protein is in the form of a homopolymer.

[0007] Furthermore, a phospholipid bilayer is constructed at the micropores, and insect olfactory receptor proteins are embedded in the phospholipid bilayer. This is obtained through the following steps: insect olfactory receptor proteins and phospholipids are mixed and incubated to obtain an insect olfactory receptor protein-phospholipid mixture, which is then coated onto the micropores using a bubble method.

[0008] Secondly, a method for preparing a nanopore sensor based on insect olfactory receptor proteins is provided, comprising the following steps:

[0009] (1) Preparation of insect olfactory receptor protein-phospholipid mixture: An equal volume of insect olfactory receptor protein solution with a concentration of 0.32 mg / ml and DphPC (diphyidoylphosphatidylcholine) phospholipid solution with a concentration of 10 mg / ml were mixed and incubated at 4 ℃ for 16 h to obtain the 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 concentrations represent the final concentrations. The solute of the DphPC phospholipid solution was DphPC, and the solvent was n-octane.

[0010] (2) Construction of nanopore sensor based on insect olfactory receptor protein: The insect olfactory receptor protein-phospholipid mixture obtained after incubation is coated on the micropore of the measurement chip to form a double membrane, thereby obtaining a nanopore sensor based on insect olfactory receptor protein.

[0011] The measurement chip comprises two chambers, each containing an independent integrated Ag / AgCl electrode for acquiring signals of current changes over time. Each chamber contains an electrolyte solution (concentration being the final concentration in the electrolyte solution): 135 mM NaCl, 5 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 10 mM MgCl2, pH 7.3. The measurement chip has micropores with a diameter of 100 µm, and a bilayer membrane is formed by coating the micropores with a mixture of insect olfactory receptor proteins and phospholipids.

[0012] When the insect olfactory receptor protein-phospholipid mixture is coated onto the measurement chip, the insect olfactory receptor protein is embedded in the phospholipid bilayer membrane, which 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.

[0013] The insect olfactory receptor protein can be assembled into a homotetrameric odor-gated ion channel; the insect olfactory receptor protein is in an open structural state under the action of the target odor ligand, and the pore size at the opening of the open structural state is about 1 nm; the nanopore is the insect olfactory receptor protein.

[0014] Furthermore, after coating the insect olfactory receptor protein-phospholipid mixture obtained after incubation onto the micropores of the measuring chip, an electrophysiological experiment can be conducted by introducing eugenol at a working concentration into an electrolyte solution to determine whether the insect olfactory receptor protein is embedded in the bilayer membrane: if a change in current is found, it indicates that the receptor protein has been embedded in the bilayer membrane.

[0015] Thirdly, 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 change in current is detected relative to the baseline current, it is determined that the air to be tested contains floral fragrance substances.

[0016] More preferably, eugenol is first added to the electrolyte solution of the nanopore sensor to prepare the 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 fragrance substance eugenol.

[0017] The insect olfactory receptor protein is a homotetrameric insect olfactory receptor protein MhOR5, which comes from a terrestrial insect called Machilis hrabei.

[0018] In some embodiments of the present invention, the following experiments were conducted:

[0019] (1) Prepare target test sample solution: Dissolve the target molecule to be detected in DMSO to prepare target molecule stock solution, and use electrolyte solution to dilute the target molecule stock solution to working concentration.

[0020] (2) Characterization of phospholipid bilayer membrane formation: The measurement chip was connected to the lipid bilayer recording device. Electrolyte solutions were added to both chambers of the measurement chip, and a phospholipid bilayer membrane was coated at the micropores of the measurement chip using a phospholipid solution or a mixture of insect olfactory receptor proteins and phospholipids. The lipid bilayer recording device was used for electrophysiological experimental measurements, which detected and recorded the signal of current change over time. When the phospholipid bilayer membrane was formed, the two chambers of the measurement chip were in an open circuit state, the output current was 0 pA, and the membrane capacitance of the phospholipid bilayer membrane was 20-50 pF (100 µm pore size measurement chip).

[0021] (3) Detection of a solution containing the target odor molecule: The target solution is added to one chamber of the measuring chip, a +50 mV voltage is applied, and the current signal changes over time before and after the addition of the target molecule is recorded under the conditions of a 5 kHz sampling frequency and a 1 kHz low-pass filter. The data is analyzed to achieve the identification of the target molecule.

[0022] The target molecule is eugenol, the concentration of the target molecule stock solution is 150 mM, and the working concentration is 5 µM.

[0023] The method of coating the micropores of the measuring chip with a phospholipid solution is used as a control experiment for coating the micropores of the measuring chip with a phospholipid mixture of insect olfactory receptor protein and phospholipid. The control experiment is used to illustrate that the change in current signal originates from the interaction between insect olfactory receptor protein and target odor molecule.

[0024] The beneficial effects of this invention are as follows:

[0025] (1) The olfactory receptors derived from insects can recognize different odor molecules, giving insects a highly sensitive olfactory perception ability. Compared with traditional artificial materials, insect olfactory receptors can detect odor molecules with high selectivity and high sensitivity.

[0026] (2) This invention creatively employs a homotetrameric insect olfactory receptor protein to construct a nanoporous sensor, and correspondingly develops a rapid and convenient embedding method suitable for homotetramers. Specifically, it utilizes the insect olfactory receptor MhOR5 derived from the cricket (Machilis hrabei), which is an odor-gated ion channel assembled in a homotetrameric form. Compared to other heteromeric insect olfactory receptors, MhOR5 consists only of odorant receptor (OR) subunits and does not contain OR co-receptor (Orco) subunits. Choosing MhOR5 to develop the sensor reduces the complexity of olfactory receptor protein preparation, thereby making the sensor construction simpler.

[0027] (3) Nanopores can achieve single-molecule level detection. Using MhOR5 as a nanopore, the natural responsiveness of MhOR5 to odor molecules can be utilized to perform single-molecule level odor detection. However, the protein nanopores commonly used in existing technologies usually require additional protein engineering or chemical modification to achieve specific functional applications.

[0028] (4) The nanopore sensor constructed in this invention is easy to operate and takes little time. It does not require any large instruments or complex data processing, and can achieve rapid identification and direct detection of target substances. Attached Figure Description

[0029] Figure 1The image shows the SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis) results of the insect olfactory receptor protein MhOR5.

[0030] Figure 2 This is a schematic diagram illustrating the construction and odor detection application of a nanopore sensor based on insect olfactory receptor proteins. Figure 2 In the diagram, A represents the construction flowchart of a nanopore sensor based on insect olfactory receptor proteins. Figure 2 B in the diagram represents the state of insect olfactory receptor proteins in the absence of target odor molecules. Figure 2 C in the diagram represents the change of current over time when the target odor molecule is absent. Figure 2 The diagram shows the state of insect olfactory receptor proteins in the presence of the target odor molecule, represented by the symbol D. Figure 2 E in the figure is a schematic diagram of the change of current over time when the target odor molecule is present;

[0031] Figure 3 A schematic diagram of a representative continuous current trajectory at +20 mV when a bilayer film is formed on a nanopore measurement chip;

[0032] Figure 4 A schematic diagram of the continuous current trajectory of a MhOR5-based nanopore sensor at +50 mV in the absence of the target odor molecule;

[0033] Figure 5 A schematic diagram of the continuous current trajectory for detecting eugenol using a MhOR5-based nanopore sensor;

[0034] Figure 6 for Frequency distribution histogram;

[0035] Figure 7 This is a schematic diagram of the continuous current trajectory of eugenol in electrophysiological detection when MhOR5 is absent. Detailed Implementation

[0036] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. 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.

[0037] Example 1: Preparation of insect olfactory receptor protein-phospholipid mixture.

[0038] The insect olfactory receptor protein used in this embodiment is MhOR5 (PDB ID: 7LIC), synthesized by DIMAR Biotechnology (Wuhan) Co., Ltd. Structural analysis of MhOR5 revealed that this insect olfactory receptor protein assembles into a homotetrameric ion channel. When binding to the target odor molecule, the top of MhOR5 changes from a hydrophobic contractile structure to form a pore with a diameter of 9.2 mm. The pores are open, allowing hydrated ions to pass through. The purification results of the MhOR5 insect olfactory receptor protein are as follows: Figure 1 As shown.

[0039] An insect olfactory receptor protein-phospholipid mixture was prepared for the subsequent construction of a sensor bilayer membrane. First, DphPC phospholipid was dissolved in n-octane (purity ≥99%) to prepare a 10 mg / ml DphPC phospholipid solution. 5 µL of the DphPC phospholipid solution was added to 5 µL of a 0.32 mg / ml MhOR5 insect olfactory receptor protein solution. After thorough mixing, the solution was incubated overnight (16 h) at 4°C to obtain the insect olfactory receptor protein-phospholipid mixture. The solvent for the insect olfactory receptor protein solution was: 150 mM sodium chloride solution, 20 mM Tris (tris(hydroxymethyl)aminomethane) buffer, pH 8.0.

[0040] Example 2: Construction of an electrophysiological detection platform.

[0041] The nanopore measurement chip was used for subsequent recombination of insect olfactory receptor proteins on a phospholipid bilayer. First, the nanopore measurement chip was washed with ultrapure water and ethanol, respectively, and then dried under nitrogen. Next, the two measurement chambers of the chip were filled with an electrolyte solution prepared from ultrapure water: 135 mM NaCl, 5 mM KCl, 2 mM MgCl2, 2 mM CaCl2, and 10 mM Hepes (4-hydroxyethylpiperazine ethanesulfonic acid) buffer, pH 7.3. The chip was then connected to an electrophysiological signal readout device (current amplifier) ​​for real-time measurement and recording of events occurring in the ion channels with ultra-low noise. The nanopore measurement chip and electrophysiological signal readout device can be the Elements BLM Chip or the fully integrated handheld portable device Nanopore Reader 100 kHz.

[0042] Example 3: Formation of phospholipid bilayer and embedding of insect olfactory receptor proteins.

[0043] By assembling a phospholipid bilayer and embedding insect olfactory receptor proteins on a nanopore measurement chip, a nanopore sensor based on insect olfactory receptor proteins was constructed for detecting target odor molecules by measuring changes in channel current. Figure 2 Using the phospholipid solution obtained in Example 1 or the incubated insect olfactory receptor protein-phospholipid mixture, a bilayer film was formed by coating the micropores of the measurement chip treated in Example 2, and the film formation status was checked. Preferably, the coating method employed was the bubble method, specifically the lipid-covered bubble method. The specific steps were as follows: the tip of a pipette was immersed in the phospholipid solution obtained in Example 1 or the incubated insect olfactory receptor protein-phospholipid mixture to collect the liquid. Then, air bubbles were generated near the micropores of the measurement chip using the pipette tip. The air bubbles contacted the micropores of the measurement chip, causing the lipid or lipid mixture covering the surface of the air bubbles to coat the micropores, forming a bilayer film. During the formation of the phospholipid bilayer film, the two chambers of the measurement chip were in an open-circuit state, and the output current was 0 pA. Figure 3 The typical membrane capacitance of a phospholipid bilayer is 20-50 pF (for a 100 µm pore size measurement chip). In the control experiment, a phospholipid solution was used to coat the bilayer. When an insect olfactory receptor protein-phospholipid mixture was used to coat the bilayer, the insect olfactory receptor protein was embedded within the phospholipid bilayer.

[0044] Example 4: Electrophysiological detection characterization of nanopore sensors based on insect olfactory receptor proteins.

[0045] The sensor prepared in Example 3 was characterized using electrophysiological measurement methods. With a voltage of +50 mV, and under sampling frequency of 5 kHz and low-pass filtering of 1 kHz, the current signal was recorded over time in the electrolyte solution environment before the addition of the target molecule. Figure 4 As shown, in the absence of the target molecule, the output current remained at 0 pA, and no channel opening event of the MhOR5 insect olfactory receptor protein was observed.

[0046] Example 5: Detection of floral scent molecules using a nanopore sensor based on insect olfactory receptor proteins.

[0047] Eugenol to be detected was dissolved in DMSO to prepare a 150 mM eugenol stock solution. This stock solution was then diluted 100-fold with the electrolyte solution prepared in Example 2. Next, the diluted eugenol solution was added to the electrolyte solution in the measurement chip chamber characterized in Example 4 to achieve a working concentration of 5 µM. Data was recorded using the same electrophysiological measurement conditions as in Example 4. Figure 5 As shown, for nanopore sensors based on insect olfactory receptor proteins, when the target odor molecule eugenol is added, the channel opening event generated by the binding of the MhOR5 insect olfactory receptor protein to eugenol can be observed. ( ) is the ion channel opening current ( ) and ion channel shut-off current ( The difference between them represents the event magnitude of the ion channel opening event, such as Figure 6 As shown (the histogram has been Gaussian fitted, and the fitted line is marked with a solid line), the ion channel current corresponding to this event increases; as... Figure 7 As shown, when insect olfactory receptor proteins are absent (as described in Example 3, only a phospholipid solution was used to construct the bilayer membrane for subsequent electrophysiological measurements), 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 a nanopore sensor based on insect olfactory receptor proteins can be applied to the detection of target odor molecules.

[0048] In summary, this invention integrates insect olfactory receptor proteins as sensitive elements into a nanopore measurement system by embedding them into a phospholipid bilayer membrane. Then, a nanopore electrophysiological measurement experiment is used to measure the current signal of the insect olfactory receptor protein in response to odor molecules. Based on the changes in the detected current signal, the target odor molecule can be detected directly and rapidly.

Claims

1. An insect olfactory receptor protein-based nanopore sensor, characterized in that, The sensor comprises two chambers filled with electrolyte solution, the two chambers are separated by a partition sheet with micropores, and each chamber is integrated with an electrode; a phospholipid bilayer is constructed at the micropores, and insect olfactory receptor proteins are embedded in the phospholipid bilayer as nanopores of the sensor; wherein the insect olfactory receptor proteins are in the form of homomultimers; the phospholipid bilayer is constructed at the micropores, and insect olfactory receptor proteins are embedded in the phospholipid bilayer by the following steps: incubating insect olfactory receptor proteins with phospholipids to obtain a mixture of insect olfactory receptor proteins and phospholipids, and coating the mixture on the micropores by the bubble method.

2. The nanopore sensor of claim 1, wherein, The concentration of the insect olfactory receptor protein solution is 0.32 mg / ml, the concentration of the phospholipid solution is 10 mg / ml, and the two are mixed in equal volumes to obtain the mixture of insect olfactory receptor proteins and phospholipids after incubation at 4 ℃ for 16 h.

3. The nanopore sensor of claim 1, wherein, The phospholipid is diarachidoyl phosphatidylcholine.

4. The nanopore sensor of claim 1, wherein, The diameter of the micropore is 100 µm.

5. The nanopore sensor of claim 1, wherein, The electrolyte solution is an aqueous alkali metal halide solution.

6. Use of the nanopore sensor according to any one of claims 1 to 5 for the detection of a floral odorant, characterized in that, Insect olfactory receptor proteins adopt a homodimeric insect olfactory receptor protein Mh OR5.

7. Use according to claim 6, characterized in that, The application specifically comprises: sampling odor molecules in the air to be tested, transferring to the electrolyte solution of the nanopore sensor, applying a voltage between the electrodes, recording the current signal, and if a current change is detected relative to the baseline current, it is judged that the air to be tested contains a floral odor substance.

8. Use according to claim 6, characterized in that, The floral odor substance is eugenol.

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