A method for preparing a biosensor for detecting geosmin based on human olfactory receptor OR51S1

Abstract

The present invention provides a method for preparing a biosensor for detecting geosmin based on the human olfactory receptor OR51S1, belonging to the technical field of biosensors, and specifically relates to the preparation of olfactory receptor cells, the preparation of nanovesicles containing OR51S1 protein, and the preparation method of a geosmin detection sensor. The sensor includes a screen-printed gold electrode, and nitrocellulose is modified on the surface of the working electrode of the screen-printed gold electrode, which contains nanovesicles, and the nanovesicles contain olfactory receptor proteins, and the olfactory receptor protein is OR51S1 protein, which is expressed by HEK-293T cells. The sensor prepared by the above method of the present invention has the characteristics of being fast, sensitive and suitable for on-site detection, effectively improving the sensitivity and operational convenience of geosmin detection.

Description

Technical Field

[0001] The present invention belongs to the field of biosensors, and particularly relates to a method for preparing a biosensor for detecting geosmin based on human olfactory receptor OR51S1. Background Art

[0002] Geosmin is mainly produced by algal metabolism and is a typical earthy-musty odor substance. The presence of geosmin not only directly causes off-flavors in drinking water and affects people's drinking experience, but also accumulates in aquatic products, reducing the quality and economic value of aquatic products. Therefore, it is also an important indicator concerned by the aquatic product industry such as fish. So, the rapid detection of geosmin is of great significance.

[0003] Currently, the common evaluation and analysis methods for odor substances can be divided into two categories: sensory analysis methods and instrumental analysis methods. Sensory analysis methods are mostly carried out by trained professional testers to evaluate the odor type and intensity, which cannot be accurately quantified, and are labor-consuming and subjective. Instrumental analysis methods usually involve the combination and coupling of water sample pretreatment methods and detection techniques: Pretreatment methods include solid-phase microextraction, liquid-liquid extraction, purge and trap, etc.; Detection techniques mainly include gas chromatography-tandem mass spectrometry (GC-MS / MS) and comprehensive two-dimensional gas chromatography-mass spectrometry (GC×GC-MS), etc. Among them, solid-phase microextraction combined with gas chromatography-tandem mass spectrometry is the main method for quantitatively detecting geosmin at present. This method has certain separation ability, can achieve a relatively low detection limit, high recovery rate and good repeatability, but has a high equipment cost, relatively complex operation and maintenance, and a long single-sample detection time, and is not suitable for on-site rapid detection and emergency treatment of water body odor events.

[0004] Therefore, there is an urgent practical need to develop a method for detecting geosmin that is rapid, accurate, low-cost and suitable for on-site detection. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for preparing a biosensor for detecting geosmin based on human olfactory receptor OR51S1.

[0006] The technical solution adopted by the present invention to achieve the above purpose is as follows:

[0007] A method for preparing a biosensor for detecting geosmin based on human olfactory receptor OR51S1, comprising:

[0008] Culturing HEK-293T cells transfected with OR51S1 expression plasmid using cell culture medium to prepare nanovesicles containing OR51S1 protein; the cell culture medium contains L-homocysteine sulfinic acid and 3-(3-acetamidophenyl)propionic acid;

[0009] The nanovesicles containing OR51S1 protein are added to the working electrode of a screen-printed gold electrode, and the working electrode is modified with a nitrocellulose layer. The nanovesicles containing OR51S1 protein are located in the nitrocellulose layer.

[0010] Preferably, the cell culture medium uses serum-free DMEM medium as a diluent.

[0011] More preferably, the mass-volume ratio of L-homocysteine sulfinic acid to serum-free DMEM medium is 3.4 - 34 μg:100 - 1000 mL; or, the mass-volume ratio of 3-(3-acetamidophenyl)propionic acid to serum-free DMEM medium is 4.2 - 42 μg:100 - 1000 mL.

[0012] Preferably, the cell culture medium also contains cytochalasin B.

[0013] More preferably, the mass-volume ratio of cytochalasin B to serum-free DMEM medium is 1 - 20 mg:100 - 1000 mL.

[0014] Preferably, the nitrocellulose layer is derived from a nitrocellulose membrane. The nitrocellulose in the nitrocellulose layer is measured by the area of the nitrocellulose membrane, and the mass-area ratio of the nanovesicles to the nitrocellulose in the nitrocellulose layer is 3.75 - 5 mg:2000 - 2500 mm 2 .

[0015] Preferably, in the preparation of the geosmin detection sensor, a nitrocellulose dispersion is dropped onto the surface of the working electrode of a screen-printed gold electrode, and then a nanovesicle solution is dropped. After standing and drying, a biosensor for detecting geosmin based on human olfactory receptor OR51S1 is obtained.

[0016] More preferably, the nitrocellulose dispersion is obtained by dispersing a nitrocellulose membrane in methanol. The pore size of the nitrocellulose membrane is 0.45 - 0.8 μm, and the area-volume ratio of the nitrocellulose membrane dispersed in methanol is 1 - 5 mm 2 :5 - 50 μL.

[0017] More preferably, the nanovesicle solution is obtained by incubating HEK-293T cells containing OR51S1 protein and then centrifuging, and resuspending with PBS containing a protease inhibitor cocktail. The volume ratio of the protease inhibitor cocktail to PBS in PBS is 1 - 2 mL:100 - 200 mL, and the mass-volume ratio of the total protein in the nanovesicles to PBS containing a protease inhibitor cocktail is 10 - 100 µg:0.1 - 1 mL.

[0018] The present invention discloses a biosensor for detecting geosmin based on human olfactory receptor OR51S1 prepared by the above method.

[0019] The present invention discloses a biosensor for detecting geosmin based on human olfactory receptor OR51S1, comprising: a screen-printed gold electrode, and nitrocellulose modified on the surface of the working electrode of the screen-printed gold electrode, which contains nanovesicles, and the nanovesicles contain olfactory receptor proteins, and the olfactory receptor protein is OR51S1 protein, expressed by HEK-293T cells;

[0020] The nanovesicles are obtained by centrifugation after incubation of HEK-293T cells containing OR51S1 protein in a serum-free DMEM medium containing cytochalasin B, L-homocysteine sulfinic acid, and 3-(3-acetamidophenyl)propionic acid. Under the synergistic effect of the culture medium additives composed of cytochalasin B, L-homocysteine sulfinic acid, and 3-(3-acetamidophenyl)propionic acid, the physiological processes of cells can be regulated, a suitable redox environment inside the cells can be maintained to ensure the quality of proteins and membrane structures inside the nanovesicles, and lipid metabolism can be participated in to regulate the composition and properties of the vesicle membrane, thereby facilitating the generation of nanovesicles containing OR51S1 protein, realizing the increase in the concentration of OR51S1 protein and the number of nanovesicles, enhancing the recognition and binding ability of the sensor to geosmin, and thus improving the sensitivity and accuracy of the sensor.

[0021] Preferably, the mass-volume ratio of cytochalasin B to the serum-free DMEM medium is 1-20 mg: 100-1000 mL.

[0022] Preferably, the mass-volume ratio of L-homocysteine sulfinic acid to the serum-free DMEM medium is 3.4-34 μg: 100-1000 mL.

[0023] Preferably, the mass-volume ratio of 3-(3-acetamidophenyl)propionic acid to the serum-free DMEM medium is 4.2-42 μg: 100-1000 mL.

[0024] Preferably, the mass-area ratio of the total protein in the nanovesicles to the nitrocellulose is 3.75-5 mg: 2000-2500 mm 2 .

[0025] The present invention provides a method for preparing a biosensor for detecting geosmin based on human olfactory receptor OR51S1. A nitrocellulose dispersion is dropped onto the surface of the working electrode of the screen-printed gold electrode, and then a nanovesicle solution is dropped. After standing and drying, a biosensor for detecting geosmin based on human olfactory receptor OR51S1 is obtained.

[0026] Preferably, the nitrocellulose dispersion is obtained by dispersing a nitrocellulose membrane in methanol. The pore size of the nitrocellulose membrane is 0.45-0.8 μm, and the area-volume ratio of the nitrocellulose membrane dispersed in methanol is 1-5 mm 2: 5 - 50 μL.

[0027] Preferably, the nanovesicle solution is obtained by centrifuging HEK - 293T cells containing OR51S1 protein after incubation and resuspending with PBS containing protease inhibitor cocktail.

[0028] Preferably, the volume ratio of protease inhibitor cocktail to PBS in PBS is 1 - 2 mL : 100 - 200 mL, and the mass - volume ratio of total protein in nanovesicles to PBS containing protease inhibitor cocktail is 10 - 100 μg : 0.1 - 1 mL.

[0029] Preferably, the expression of OR51S1 protein is achieved by co - transfecting OR51S1 expression plasmid, receptor trafficking protein RTP1, and receptor expression enhancing protein REEP into HEK - 293T cells using Lipofectamine 2000 transfection reagent.

[0030] More preferably, cocamidopropyl dimethylamine lactate can be added in the preparation of nanovesicles containing OR51S1 protein. The mass - volume ratio of cocamidopropyl dimethylamine lactate to serum - free DMEM medium is 0.1 - 1 mg : 100 - 1000 mL. The addition of cocamidopropyl dimethylamine lactate enables the nanovesicles to disperse better in the solution during the incubation of nanovesicles containing OR51S1 protein, which is more conducive to long - term storage under freezing conditions and ensures that its activity and function are not affected, maintaining the stability of the system. At the same time, it optimizes the interaction between nanovesicles and detection components such as screen - printed gold electrodes, enabling the nanovesicles to better specifically bind to geosmin and improving the detection sensitivity and accuracy of the biosensor.

[0031] The present invention also provides a method for preparing an olfactory receptor, including:

[0032] Selecting OR51S1 as the human olfactory receptor for geosmin and HEK - 293T cells as the host cells. Obtaining the gene sequence information of the corresponding olfactory receptor OR51S1 for geosmin from the Olfactory Receptor DataBase and chemically synthesizing the gene artificially; cloning the OR51S1 gene between the restriction enzyme cleavage sites MluI and NotI of the mammalian eukaryotic expression vector pCI to obtain the OR51S1 expression plasmid; using Lipofectamine 2000 transfection reagent to transfect the OR51S1 expression plasmid, receptor trafficking protein RTP1, and receptor expression enhancing protein REEP into HEK - 293T cells to express OR51S1 protein, and the plasmid transfection duration is 24 - 48 h.

[0033] The present invention also provides a method for preparing nanovesicles containing OR51S1 protein, including:

[0034] Dissolve L-homocysteine sulfinic acid in deionized water to obtain an L-homocysteine sulfinic acid solution; dissolve 3-(3-acetamidophenyl)propionic acid in DMSO to obtain a 3-(3-acetamidophenyl)propionic acid solution; add cytochalasin B, the L-homocysteine sulfinic acid solution and the 3-(3-acetamidophenyl)propionic acid solution to a serum-free DMEM medium, and incubate HEK293T cells at 36-37 °C and 4-5% CO2. After incubating for 30-60 min, centrifuge the mixture to remove the remaining cells, centrifuge the supernatant to obtain a nanovesicle precipitate; resuspend with PBS containing a protease inhibitor cocktail to obtain nanovesicles containing the OR51S1 protein. Use a Qubit protein assay kit to quantify the total protein concentration, dilute with PBS containing a protease inhibitor cocktail to obtain a nanovesicle solution, and store frozen.

[0035] Preferably, in the L-homocysteine sulfinic acid solution, the mass-volume ratio of L-homocysteine sulfinic acid to deionized water is 0.17-1.7 mg: 1-10 mL.

[0036] Preferably, in the 3-(3-acetamidophenyl)propionic acid solution, the mass-volume ratio of 3-(3-acetamidophenyl)propionic acid to DMSO is 0.21-2.1 mg: 1-10 mL.

[0037] Preferably, the mass-volume ratio of cytochalasin B to the serum-free DMEM medium is 1-20 mg: 100-1000 mL.

[0038] Preferably, in terms of the mass of L-homocysteine sulfinic acid, the mass-volume ratio of the L-homocysteine sulfinic acid solution to the serum-free DMEM medium is 3.4-34 μg: 100-1000 mL.

[0039] Preferably, in terms of the mass of 3-(3-acetamidophenyl)propionic acid, the mass-volume ratio of 3-(3-acetamidophenyl)propionic acid to the serum-free DMEM medium is 4.2-42 μg: 100-1000 mL.

[0040] Preferably, the volume ratio of the protease inhibitor cocktail to PBS in PBS is 1-2: 100-200, and the mass-volume ratio of the total protein in the nanovesicles to PBS containing the protease inhibitor cocktail is 10-100 μg: 0.1-1 mL.

[0041] Preferably, the centrifugation speed of the mixture is 300-700 × g for 5-15 min. The centrifugation speed of the supernatant is 13000-17000 × g for 20-40 min.

[0042] The present invention also provides a method for preparing a geosmin detection sensor, comprising: dispersing a nitrocellulose membrane in methanol, and vortexing for 5 - 15 min at room temperature to fully dissolve to obtain a nitrocellulose dispersion; dropping the nitrocellulose dispersion onto the surface of a working electrode, allowing it to stand at room temperature until the methanol volatilizes and dries, and then dropping an OR51S1 nanovesicle solution onto the working electrode, and allowing it to stand at room temperature for 1 - 3 h to obtain the geosmin detection sensor.

[0043] Preferably, the pore size of the nitrocellulose membrane is 0.45 - 0.8 μm, and the area - volume ratio of the nitrocellulose membrane dispersed in methanol is 1 - 5 mm 2 : 5 - 50 μL.

[0044] Preferably, the volume ratio of the nitrocellulose dispersion to the nanovesicle solution is 2 - 10:7.5 - 37.5.

[0045] Preferably, the volume - area ratio of the nanovesicle solution to the working electrode of the screen - printed gold electrode is 7.5 - 37.5 μL:10 - 50 mm 2 .

[0046] The present invention discloses a biosensor prepared by the above - mentioned method.

[0047] Since the present invention constructs a heterologous expression system capable of highly expressing an olfactory receptor protein with specific recognition ability for geosmin, and provides a method for preparing olfactory receptor cell nanovesicles. The cell culture is optimized by adding L - homocysteine sulfinic acid, 3 - (3 - acetamidophenyl) propionic acid and cocamidopropyl dimethylamine lactate, thus having the following beneficial effects: the protein expression level of the nanovesicles containing the OR51S1 protein is high, and the number of nanovesicles is large. Therefore, the present invention is a biosensor with high sensitivity, rapidity and portability and its preparation method. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 It is a schematic diagram of the test result of the number of nanovesicles containing the OR51S1 protein.

[0049] Figure 2 It is a schematic diagram of the test result of the protein expression level of the nanovesicles containing the OR51S1 protein.

[0050] Figure 3 It is a schematic diagram of the cyclic voltammetry curve of geosmin - OR51S1. DETAILED DESCRIPTION OF THE INVENTION

[0051] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0052] First, the concepts involved in the present application will be described below with reference to the accompanying drawings. It should be noted here that the following descriptions of each concept are only for making the content of the present application easier to understand and do not represent a limitation on the protection scope of the present application; at the same time, without conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present application will be described in detail below with reference to the drawings and in combination with the embodiments.

[0053] Embodiment 1:

[0054] Preparation of olfactory receptor cells: Select OR51S1 as the human olfactory receptor for geosmin, and HEK-293T cells as the host cells; obtain the gene sequence information of the olfactory receptor OR51S1 corresponding to geosmin from the Olfactory Receptor DataBase, and chemically synthesize this gene artificially; clone the OR51S1 gene between the restriction enzyme sites MluI and NotI of the mammalian eukaryotic expression vector pCI to obtain the OR51S1 expression plasmid; use Lipofectamine2000 transfection reagent to transfect the OR51S1 expression plasmid, receptor transport protein RTP1, and receptor expression enhancer protein REEP into HEK-293T cells to express the OR51S1 protein, and the plasmid transfection duration is 24h.

[0055] Preparation of nanovesicles containing OR51S1 protein: Dissolve 1.7 mg of L-homocysteine sulfinic acid in 10 mL of deionized water to obtain an L-homocysteine sulfinic acid solution; dissolve 2.1 mg of 3-(3-acetamidophenyl)propionic acid in 10 mL of DMSO to obtain a 3-(3-acetamidophenyl)propionic acid solution; add 1 mg of cytochalasin B, 100 μL of the L-homocysteine sulfinic acid solution and 100 μL of the 3-(3-acetamidophenyl)propionic acid solution to 500 mL of serum-free DMEM medium, incubate HEK293T cells, mix on a shaker for 30 min at 37 °C and 5% CO2, centrifuge the mixture at 500×g for 10 min to remove the remaining cells, take the supernatant and centrifuge at 15000×g for 30 min to obtain a nanovesicle precipitate; discard the supernatant, resuspend the precipitate with PBS containing 1% protease inhibitor cocktail; quantitatively determine the total protein concentration using a Qubit protein detection kit, dilute it to 100 μg / mL with PBS containing 1% protease inhibitor cocktail, and store the nanovesicles at -80 °C.

[0056] Preparation of the sensor: Dissolve the nitrocellulose membrane in methanol and vortex for 10 min at room temperature to fully dissolve it to obtain a nitrocellulose dispersion; add 4 μL of the nitrocellulose dispersion dropwise onto the surface of the working electrode, and after standing at room temperature until the methanol evaporates and dries, add 15 μL of a 100 μg / mL OR51S1 nanovesicle solution dropwise onto the working electrode and let it stand at room temperature for 2 h. The pore size of the nitrocellulose membrane is 0.45 μm, and the area-to-volume ratio of the nitrocellulose membrane added to methanol is 1 mm²:5 μL; the area of the working electrode on the surface of the screen-printed gold electrode is 20 mm 2 . When the nitrocellulose dispersion is dropped onto the working electrode on the surface of the screen-printed gold electrode, it is required to only cover the working electrode and not contact the counter electrode and the reference electrode; when dropping the nanovesicle solution, it is required to only cover the working electrode and not contact the counter electrode and the reference electrode.

[0057] Example 2: This example is different from Example 1 only in the preparation of nanovesicles containing OR51S1 protein.

[0058] Preparation of nanovesicles containing OR51S1 protein: Dissolve 1.7 mg of L-homocysteine sulfinic acid in 10 mL of deionized water to obtain an L-homocysteine sulfinic acid solution; dissolve 2.1 mg of 3-(3-acetamidophenyl)propionic acid in 10 mL of DMSO to obtain a 3-(3-acetamidophenyl)propionic acid solution; add 1 mg of cytochalasin B, 200 μL of the L-homocysteine sulfinic acid solution, and 100 μL of the 3-(3-acetamidophenyl)propionic acid solution to 500 mL of serum-free DMEM medium, incubate HEK293T cells, mix on a shaker for 30 min at 37 °C and 5% CO2, centrifuge the mixture at 500×g for 10 min to remove residual cells, take the supernatant and centrifuge it at 15000×g for 30 min to obtain a nanovesicle precipitate; discard the supernatant, and resuspend the precipitate with PBS containing 1% protease inhibitor cocktail. Quantify the total protein concentration using a Qubit protein assay kit, dilute it to 100 μg / mL with PBS containing 1% protease inhibitor cocktail, and store the nanovesicles at -80 °C.

[0059] Example 3: This example is different from Example 1 only in the preparation of nanovesicles containing OR51S1 protein.

[0060] Preparation of nanovesicles containing OR51S1 protein: Dissolve 1.7 mg of L-homocysteine sulfinic acid in 10 mL of deionized water to obtain an L-homocysteine sulfinic acid solution; dissolve 2.1 mg of 3-(3-acetamidophenyl)propionic acid in 10 mL of DMSO to obtain a 3-(3-acetamidophenyl)propionic acid solution; dissolve 50 mg of cocamidopropyl dimethylamine lactate in 10 mL of deionized water to obtain a cocamidopropyl dimethylamine lactate solution; add 1 mg of cytochalasin B, 100 μL of the L-homocysteine sulfinic acid solution, 100 μL of the 3-(3-acetamidophenyl)propionic acid solution, and 100 μL of the cocamidopropyl dimethylamine lactate solution to 500 mL of serum-free DMEM medium, incubate HEK293T cells, mix on a shaker for 30 min at 37 °C and 5% CO2, centrifuge the mixture at 500×g for 10 min to remove residual cells, take the supernatant and centrifuge it at 15000×g for 30 min to obtain a nanovesicle precipitate; discard the supernatant, and resuspend the precipitate with PBS containing 1% protease inhibitor cocktail. Quantify the total protein concentration using a Qubit protein assay kit, dilute it to 100 μg / mL with PBS containing 1% protease inhibitor cocktail, and store the nanovesicles at -80 °C.

[0061] Example 4: This example is different from Example 1 only in the preparation of nanovesicles containing OR51S1 protein.

[0062] Preparation of nanovesicles containing OR51S1 protein: Dissolve 1.7 mg of L-homocysteine sulfinic acid in 10 mL of deionized water to obtain an L-homocysteine sulfinic acid solution; dissolve 2.1 mg of 3-(3-acetamidophenyl)propionic acid in 10 mL of DMSO to obtain a 3-(3-acetamidophenyl)propionic acid solution; dissolve 50 mg of cocamidopropyl dimethylamine lactate in 10 mL of deionized water to obtain a cocamidopropyl dimethylamine lactate solution; add 1 mg of cytochalasin B, 100 μL of the L-homocysteine sulfinic acid solution, 100 μL of the 3-(3-acetamidophenyl)propionic acid solution, and 200 μL of the cocamidopropyl dimethylamine lactate solution to 500 mL of serum-free DMEM medium, incubate HEK293T cells, mix on a shaker at 37 °C and 5% CO2 for 30 min, centrifuge the mixture at 500×g for 10 min to remove residual cells, take the supernatant and centrifuge at 15000×g for 30 min to obtain a nanovesicle precipitate; discard the supernatant, and resuspend the precipitate with PBS containing 1% protease inhibitor cocktail. Quantify the total protein concentration using a Qubit protein assay kit, dilute it to 100 μg / mL with PBS containing 1% protease inhibitor cocktail, and store the nanovesicles at -80 °C.

[0063] Comparative Example 1:

[0064] The difference between this comparative example and Example 1 is only that L-homocysteine sulfinic acid was not used in the preparation of the nanovesicles containing OR51S1 protein.

[0065] Comparative Example 2:

[0066] The difference between this comparative example and Example 1 is only that 3-(3-acetamidophenyl)propionic acid was not used in the preparation of the nanovesicles containing OR51S1 protein.

[0067] Comparative Example 3:

[0068] The difference between this comparative example and Example 1 is only that neither L-homocysteine sulfinic acid nor 3-(3-acetamidophenyl)propionic acid was used in the preparation of the nanovesicles containing OR51S1 protein.

[0069] Test Example 1: Determination of the number of nanovesicles

[0070] Test samples: Undiluted nanovesicles containing OR51S1 protein prepared by the methods of each example and comparative example.

[0071] Test method: Dilute the nanovesicles containing OR51S1 protein with PBS containing 1% protease inhibitor cocktail to 10 7 -10 8 mL -1Upper concentration, quantitative and particle size distribution analysis of nanovesicles were carried out using nanoparticle tracking analysis (NTA) technology. NTA provides the concentration of nanovesicles (number of particles / mL) by tracking the Brownian motion of nanovesicles in the sample and combining Stokes' law, and calculates the number of vesicles.

[0072] The test results of the number of nanovesicles containing OR51S1 protein prepared in the present invention are as Figure 1 shown. The number of nanovesicles in Example 1 is 5.1×10 8 per mL, the number of nanovesicles in Example 2 is 5.0×10 8 per mL, the number of nanovesicles in Example 3 is 7.2×10 8 per mL, and the number of nanovesicles in Example 4 is 7.1×10 8 per mL. It can be seen from Example 1 to Example 3 that in the presence of L-homocysteine sulfinic acid and 3-(3-acetamidophenyl)propionic acid, cells can release a certain number of nanovesicles according to the normal physiological process, and the dosages of various substances are in a relatively balanced state, providing a more suitable intracellular environment for the formation of nanovesicles. At the same time, the addition of cocamidopropyl dimethylamine lactate has a positive effect on the formation or stability of nanovesicles. It may interact with the nanovesicle membrane, changing the surface properties or stability of the nanovesicles, thereby facilitating the formation of nanovesicles or reducing the loss of nanovesicles during the preparation process, resulting in an increase in the detectable number of nanovesicles. In Comparative Example 1, L-homocysteine sulfinic acid was not used, and the number of vesicles decreased sharply to 1.6×10 8 per mL, fully demonstrating the indispensability of L-homocysteine sulfinic acid in the vesicle formation process, and its absence greatly inhibits vesicle generation. In Comparative Example 2, 3-(3-acetamidophenyl)propionic acid was not used, and the number of vesicles was 2.0×10 8 per mL, which was also significantly lower than that in Example 1, proving that 3-(3-acetamidophenyl)propionic acid plays an important role in vesicle formation. In Comparative Example 3, both L-homocysteine sulfinic acid and 3-(3-acetamidophenyl)propionic acid were not used, and the number of vesicles was only 0.7×10 8 per mL, indicating that these two substances together constitute the key basis for vesicle formation.

[0073] The number of nanovesicles containing OR51S1 protein prepared in the present invention is in the range of 5.0 - 7.0×10 8 per mL, significantly enhancing the number of nanovesicles, improving the recognition and binding ability of the sensor to geosmin, and thus improving the sensitivity and accuracy of the sensor.

[0074] Test Example 2: Determination of protein expression level

[0075] Test samples: Undiluted nanovesicles containing OR51S1 protein prepared by the methods of each example and comparative example.

[0076] Test method: Dilute the nanovesicles containing OR51S1 protein with PBS containing 1% protease inhibitor cocktail, and quantify the total protein concentration using a Qubit protein assay kit.

[0077] The test results of the protein expression level of the nanovesicles containing OR51S1 protein prepared in the present invention are as Figure 2 shown. The protein concentration in Example 1 is 525 µg / mL, the protein concentration in Example 2 is 564 µg / mL, the protein concentration in Example 3 is 707 µg / mL, and the protein concentration in Example 4 is 735 µg / mL. From Example 1 to Example 2, it can be seen that the change in the dosage of L-homocysteine sulfinic acid mainly affects the total protein concentration, causing a slight increase, indicating that this substance is involved in the intracellular protein synthesis-related process to a certain extent and helps to increase the protein content in the vesicles. In Example 3, on the basis of Example 1, cocamidopropyl dimethylamine lactate is added, and the total protein concentration increases significantly, showing that the addition of cocamidopropyl dimethylamine lactate has a positive effect on the total protein concentration. It may promote protein synthesis and stability by interacting with intracellular proteins, enabling more proteins to be effectively integrated into the vesicles, thus achieving an increase in the total protein concentration. In Example 4, when the concentration of cocamidopropyl dimethylamine lactate is increased to 10 µg / mL, the total protein concentration reaches the highest, reflecting the positive effect of cocamidopropyl dimethylamine lactate on protein synthesis. In Comparative Example 1, L-homocysteine sulfinic acid is not used, and the total protein concentration is only 380 mg / mL, demonstrating the key role of L-homocysteine sulfinic acid in intracellular physiological processes. Its absence leads to a significant decrease in the total protein concentration and plays a crucial role in promoting the protein synthesis process. In Comparative Example 2, 3-(3-acetamidophenyl)propionic acid is not used, and the total protein concentration is 423 mg / mL, proving the importance of 3-(3-acetamidophenyl)propionic acid for protein synthesis and vesicle formation. Its absence will significantly decrease the total protein concentration and the number of vesicles. In Comparative Example 3, both L-homocysteine sulfinic acid and 3-(3-acetamidophenyl)propionic acid are not used, and the total protein concentration is as low as 356 mg / mL. The common absence of these two substances results in an extremely low total protein concentration, further emphasizing that they jointly constitute the key basis for intracellular protein synthesis and are indispensable. The total protein concentration of the nanovesicles containing OR51S1 protein prepared in the present invention is in the range of 525 - 735 µg / mL. This relatively stable and high-level total protein concentration significantly enhances the performance of the nanovesicles, greatly improving the recognition and binding ability of the sensor to geosmin, thereby effectively improving the sensitivity and accuracy of the sensor.

[0078] Schematic diagram of the cyclic voltammetry curve of geosmin-OR51S1 is as follows Figure 3 As shown, a biosensor for detecting geosmin in aqueous solution using the human olfactory receptor OR51S1 prepared in Example 1 includes the following steps: Prepare a redox couple solution containing 5 mmol / L potassium ferricyanide, 5 mmol / L potassium ferrocyanide, and 0.1 mol / L potassium chloride with deionized water. Dilute the geosmin methanol standard solution with deionized water to obtain geosmin test solutions with concentrations of 10 ng / L, 100 ng / L, 1 μg / L, 10 μg / L, 100 μg / L, and 1 mg / L. Additionally, set 0 ng / L as a blank control; Mix the test solution and the redox couple solution in equal volumes to obtain a mixed test solution; Connect the working electrode, counter electrode, and reference electrode of the microelectrode sensor modified with OR51S1 nanovesicles to the electrochemical workstation one by one through an adapter and wires. Drop 200 μL of the mixed test solution onto the surface of the microelectrode to ensure that all three electrodes are completely immersed. Measure the reduction peak current using cyclic voltammetry. The parameters of cyclic voltammetry are set as follows: scanning speed 50 mV / s, scanning range -0.3 V to 0.7 V, and the number of scanning cycles 2 - 3 cycles.

[0079] The above-described embodiments and / or implementation manners are only used to illustrate the preferred embodiments and / or implementation manners for realizing the technology of the present invention, and do not impose any formal restrictions on the implementation manners of the technology of the present invention. Any person skilled in the art, without departing from the scope of the technical means disclosed in the content of the present invention, may make some modifications or changes to other equivalent embodiments, but should still be regarded as the same technology or embodiment as the present invention in essence.

[0080] In this article, specific examples are used to elaborate on the principles and implementation manners of the present application. The description of the above embodiments is only used to help understand the method and its core idea of the present application. The above are only the preferred implementation manners of the present application. It should be noted that due to the limited nature of written expression and objectively existing infinite specific structures, for those of ordinary skill in the art, without departing from the principles of the present application, several improvements, refinements, or changes can be made, or the above technical features can be combined in an appropriate manner; these improvements, refinements, changes, or combinations, or directly applying the concept and technical solution of the invention to other occasions without improvement, should all be regarded as the protection scope of the present application.

Claims

1. A method for preparing a biosensor for detecting geosmin based on human olfactory receptor OR51S1, comprising: HEK-293T cells transfected with an OR51S1 expression plasmid are cultured in a cell culture medium to prepare nanovesicles containing the OR51S1 protein; the cell culture medium contains L-homocysteinesulfinic acid and 3-(3-acetylaminophenyl)propionic acid; Adding the nanovesicles containing the OR51S1 protein to a working electrode of a screen-printed gold electrode, wherein the working electrode is modified with a nitrocellulose layer, and the nanovesicles containing the OR51S1 protein are located in the nitrocellulose layer; The cell culture fluid uses serum-free DMEM medium as a diluent, and the mass volume ratio of L-homocysteinesulfinic acid to serum-free DMEM medium is 3.4-34 μg:100-1000 mL; or, the mass volume ratio of 3-(3-acetylaminophenyl)propionic acid to serum-free DMEM medium is 4.2-42 μg:100-1000 mL; The cell culture fluid also contains cytochalasin B, and the mass volume ratio of the cytochalasin B to the serum-free DMEM culture medium is 1-20 mg:100-1000 mL.

2. The method for preparing a biosensor for detecting geosmin based on human olfactory receptor OR51S1 according to claim 1, characterized in that: The nitrocellulose layer is derived from a nitrocellulose membrane, the nitrocellulose in the nitrocellulose layer is measured by the area of ​​the nitrocellulose membrane, and the mass area ratio of the nanovesicles to the nitrocellulose in the nitrocellulose layer is 3.75-5 mg:2000-2500 mm 2 .

3. The method for preparing a biosensor for detecting geosmin based on human olfactory receptor OR51S1 according to claim 1, characterized in that: In the preparation of the geosmin detection sensor, a nitrocellulose dispersion is added dropwise onto the working electrode surface of the screen-printed gold electrode, and then a nanovesicle solution is added dropwise. After standing and drying, a biosensor for detecting geosmin based on the human olfactory receptor OR51S1 is obtained.

4. The method for preparing a biosensor for detecting geosmin based on human olfactory receptor OR51S1 according to claim 3, characterized in that: The nitrocellulose dispersion is obtained by dispersing a nitrocellulose membrane in methanol, the pore size of the nitrocellulose membrane is 0.45-0.8 μm, and the area-to-volume ratio of the nitrocellulose membrane dispersed in methanol is 1-5 mm 2 :5-50μL.

5. The method for preparing a biosensor for detecting geosmin based on human olfactory receptor OR51S1 according to claim 3, characterized in that: The nanovesicle solution is obtained by incubating HEK-293T cells containing OR51S1 protein and then centrifuging them, and resuspending them in PBS containing a protease inhibitor cocktail, wherein the volume ratio of the protease inhibitor cocktail to the PBS is 1-2 mL:100-200 mL, and the mass volume ratio of the total protein in the nanovesicles to the PBS containing the protease inhibitor cocktail is 10-100 µg:0.1-1 mL.

6. A biosensor for detecting geosmin based on human olfactory receptor OR51S1 prepared by the method according to any one of claims 1 to 5.

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