Preparation method of biosensor for detecting geosmin based on human olfactory receptor OR51S1

Abstract

The invention provides a preparation method of a biosensor for detecting geosmin based on a human olfactory receptor OR51S1, belongs to the technical field of biosensors, and particularly relates to preparation of olfactory receptor cells, preparation of nano-vesicles containing OR51S1 protein and a preparation method of a geosmin detection sensor. The sensor comprises a silk-screen printing gold electrode and nitrocellulose modified on the surface of a working electrode of the silk-screen printing gold electrode, the nitrocellulose contains nano-vesicles, the nano-vesicles contain olfactory receptor protein, and the olfactory receptor protein is OR51S1 protein and is expressed by HEK-293T cells. The sensor prepared by the method has the characteristics of rapidness, sensitivity and suitability for field detection, and the sensitivity and the operation convenience of geosmin detection are effectively improved.

Description

Technical Field

[0001] The invention belongs to the field of biosensors, and in particular 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 algae metabolism and is a typical earthy and musty substance. The presence of geosmin not only directly causes the odor of drinking water, affecting people's drinking experience, but also accumulates in aquatic products, which will reduce the quality and economic value of aquatic products. Therefore, it is also an important indicator of concern in the fish and other aquatic product industries, so the rapid detection of geosmin is of great significance.

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

[0004] Therefore, there is an urgent need to develop a rapid, accurate, low-cost and field-suitable method for detecting geosmin. Summary of the invention

[0005] The object 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-mentioned purpose is: 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 were cultured in a cell culture medium to prepare nanovesicles containing the OR51S1 protein; the cell culture medium contained L-homocysteinesulfinic acid and 3-(3-acetylaminophenyl)propionic acid; The nanovesicles containing OR51S1 protein were added to the working electrode of the screen-printed gold electrode, which was modified with a nitrocellulose layer, and the nanovesicles containing OR51S1 protein were located in the nitrocellulose layer.

[0007] Preferably, serum-free DMEM medium is used as the diluent in the cell culture medium.

[0008] More preferably, 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.

[0009] Preferably, the cell culture medium also contains cytochalasin B.

[0010] More preferably, the mass volume ratio of cytochalasin B to serum-free DMEM medium is 1-20 mg: 100-1000 mL.

[0011] 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 .

[0012] Preferably, in the preparation of the geosmin detection sensor, the nitrocellulose dispersion is dripped onto the working electrode surface of the screen-printed gold electrode, and then the nanovesicle solution is dripped. After standing and drying, a biosensor for detecting geosmin based on the human olfactory receptor OR51S1 is obtained.

[0013] 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-to-volume ratio of the nitrocellulose membrane dispersed in methanol is 1-5 mm 2 :5-50μL.

[0014] 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, wherein the volume ratio of the protease inhibitor cocktail to PBS in 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.

[0015] The invention discloses a biosensor for detecting geosmin based on human olfactory receptor OR51S1, which is prepared by the above method.

[0016] 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 working electrode surface of the screen-printed gold electrode, wherein the nitrocellulose contains nanovesicles, wherein the nanovesicles contain olfactory receptor proteins, and the olfactory receptor proteins are OR51S1 proteins expressed by HEK-293T cells; The nanovesicles were obtained by centrifugation after incubation of HEK-293T cells containing OR51S1 protein in serum-free DMEM medium containing cytochalasin B, L-homocysteine ​​sulfinic acid and 3-(3-acetylaminophenyl) propionic acid. Under the synergistic effect of the culture medium additives composed of cytochalasin B, L-homocysteine ​​sulfinic acid and 3-(3-acetylaminophenyl) propionic acid, the cell physiological process can be regulated, the appropriate redox environment in the cell can be maintained to ensure the quality of the protein and membrane structure in the nanovesicles, and the composition and properties of the vesicle membrane can be regulated by participating in lipid metabolism, thereby facilitating the generation of nanovesicles containing OR51S1 protein, achieving an increase in the concentration of OR51S1 protein and the number of nanovesicles, and enhancing the sensor's recognition and binding ability to geosmin, thereby improving the sensitivity and accuracy of the sensor.

[0017] Preferably, the mass volume ratio of cytochalasin B to serum-free DMEM medium is 1-20 mg: 100-1000 mL.

[0018] Preferably, the mass volume ratio of L-homocysteinesulfinic acid to serum-free DMEM medium is 3.4-34 μg:100-1000 mL.

[0019] Preferably, the mass volume ratio of 3-(3-acetylaminophenyl)propionic acid to serum-free DMEM medium is 4.2-42 μg:100-1000 mL.

[0020] Preferably, the mass area ratio of total protein to nitrocellulose in the nanovesicles is 3.75-5 mg: 2000-2500 mm 2 .

[0021] The present invention provides a method for preparing a biosensor for detecting geosmin based on human olfactory receptor OR51S1. A nitrocellulose dispersion is dripped onto the working electrode surface of a screen-printed gold electrode, and then a nanovesicle solution is dripped. After standing and drying, a biosensor for detecting geosmin based on human olfactory receptor OR51S1 is obtained.

[0022] 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-to-volume ratio of the nitrocellulose membrane dispersed in methanol is 1-5 mm 2 :5-50μL.

[0023] Preferably, the nanovesicle solution is obtained by incubating HEK-293T cells containing OR51S1 protein, centrifuging the cells, and resuspending the cells in PBS containing a protease inhibitor cocktail.

[0024] Preferably, 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 the PBS containing the protease inhibitor cocktail is 10-100 µg:0.1-1 mL.

[0025] Preferably, the expression of OR51S1 protein is achieved by co-transfecting OR51S1 expression plasmid, receptor transport protein RTP1 and receptor expression enhancing protein REEP into HEK-293T cells using Lipofectamine 2000 transfection reagent.

[0026] More preferably, cocamidopropyl dimethylamine lactate can be added to the preparation of nanovesicles containing OR51S1 protein, and the mass volume ratio of cocamidopropyl dimethylamine lactate to serum-free DMEM medium is 0.1-1mg:100-1000mL. The addition of cocamidopropyl dimethylamine lactate, in the incubation of nanovesicles containing OR51S1 protein, the nanovesicles can be better dispersed in the solution, which is more conducive to long-term storage under freezing conditions and ensuring that its activity and function are not affected, maintaining the stability of the system, and optimizing the interaction between the nanovesicles and the detection components such as the screen-printed gold electrode, so that the nanovesicles can better bind to the specificity of geosmin, and improve the detection sensitivity and accuracy of the biosensor.

[0027] The present invention also provides a method for preparing an olfactory receptor, comprising: OR51S1 was selected as the human olfactory receptor of geosmin, and HEK-293T cells were used as host cells. The gene sequence information of the olfactory receptor OR51S1 corresponding to geosmin was obtained from the Olfactory Receptor Database, and the gene was artificially synthesized by chemical method; the OR51S1 gene was cloned between the restriction sites MluI and NotI of the mammalian eukaryotic expression vector pCI to obtain the OR51S1 expression plasmid; the OR51S1 expression plasmid, receptor transport protein RTP1 and receptor expression enhancing protein REEP were transfected into HEK-293T cells using Lipofectamine2000 transfection reagent to express OR51S1 protein, and the plasmid transfection time was 24-48h.

[0028] The present invention also provides a method for preparing nanovesicles containing OR51S1 protein, comprising: L-homocysteine ​​sulfinic acid was dissolved in deionized water to obtain L-homocysteine ​​sulfinic acid solution; 3-(3-acetylaminophenyl) propionic acid was dissolved in DMSO to obtain 3-(3-acetylaminophenyl) propionic acid solution; cytochalasin B, L-homocysteine ​​sulfinic acid solution and 3-(3-acetylaminophenyl) propionic acid solution were added to serum-free DMEM medium, HEK293T cells were incubated at 36-37°C and 4-5% CO2, and the mixture was centrifuged to remove the remaining cells after incubation for 30-60 minutes, and the supernatant was centrifuged to obtain nanovesicle precipitation; resuspended with PBS containing protease inhibitor cocktail to obtain nanovesicles containing OR51S1 protein. The total protein concentration was quantified using the Qubit protein detection kit, and the nanovesicle solution was diluted with PBS containing protease inhibitor cocktail and stored frozen.

[0029] Preferably, in the L-homocysteinesulfinic acid solution, the mass volume ratio of L-homocysteinesulfinic acid to deionized water is 0.17-1.7 mg: 1-10 mL.

[0030] Preferably, in the 3-(3-acetylaminophenyl)propionic acid solution, the mass volume ratio of 3-(3-acetylaminophenyl)propionic acid to DMSO is 0.21-2.1 mg: 1-10 mL.

[0031] Preferably, the mass volume ratio of cytochalasin B to serum-free DMEM medium is 1-20 mg: 100-1000 mL.

[0032] Preferably, the L-homocysteine ​​sulfinic acid solution is measured by the mass of L-homocysteine ​​sulfinic acid, and 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.

[0033] Preferably, the 3-(3-acetylaminophenyl)propionic acid solution is measured by the mass of 3-(3-acetylaminophenyl)propionic acid, and the mass volume ratio of 3-(3-acetylaminophenyl)propionic acid to serum-free DMEM medium is 4.2-42 μg:100-1000 mL.

[0034] 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 the PBS containing the protease inhibitor cocktail is 10-100 µg:0.1-1 mL.

[0035] Preferably, the mixed solution is centrifuged at a speed of 300-700×g for 5-15 min, and the supernatant is centrifuged at a speed of 13000-17000×g for 20-40 min.

[0036] The present invention also provides a method for preparing a geosmin detection sensor, comprising: dispersing a nitrocellulose membrane in methanol, vortexing for 5-15 minutes at room temperature to fully dissolve the nitrocellulose dispersion; dropping the nitrocellulose dispersion onto the surface of a working electrode, leaving it to stand at room temperature until the methanol evaporates and dries, dropping an OR51S1 nanovesicle solution onto the working electrode, and leaving it to stand at room temperature for 1-3 hours to obtain the geosmin detection sensor.

[0037] Preferably, 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.

[0038] Preferably, the volume ratio of the nitrocellulose dispersion to the nanovesicle solution is 2-10:7.5-37.5.

[0039] 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 .

[0040] The invention discloses a biosensor prepared by the method.

[0041] The present invention provides a method for preparing olfactory receptor cell nanovesicles by constructing a heterologous expression system capable of efficiently expressing an olfactory receptor protein with specific recognition ability for geosmin. The cell culture is optimized by adding L-homocysteinesulfinic acid, 3-(3-acetylaminophenyl) propionic acid and cocamidopropyl dimethylamine lactate, thereby having the following beneficial effects: the protein expression amount of the nanovesicle containing OR51S1 protein is high and the number of nanovesicles is large. Therefore, the present invention is a highly sensitive, rapid and portable biosensor and a preparation method thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 Schematic diagram of the test results of the number of nanovesicles containing OR51S1 protein.

[0043] Figure 2 Schematic diagram of the protein expression test results of nanovesicles containing OR51S1 protein.

[0044] Figure 3 Schematic diagram of the cyclic voltammetry curve of geosmin-OR51S1. DETAILED DESCRIPTION

[0045] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. 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 creative work are within the scope of protection of the present invention.

[0046] The following first describes the concepts involved in the present application in conjunction with the accompanying drawings. It should be noted that the following description of each concept is only to make the content of the present application easier to understand, and does not limit the scope of protection of the present application; at the same time, the embodiments and features in the embodiments of the present application can be combined with each other in the absence of conflict. The present application will be described in detail with reference to the accompanying drawings and in conjunction with the embodiments.

[0047] Embodiment 1: Preparation of olfactory receptor cells: OR51S1 was selected as the human olfactory receptor for geosmin, and HEK-293T cells were used as host cells; the gene sequence information of the olfactory receptor OR51S1 corresponding to geosmin was obtained from the Olfactory Receptor Database (Olfactory Receptor DataBase), and the gene was artificially synthesized by chemical method; the OR51S1 gene was cloned between the restriction sites MluI and NotI of the mammalian eukaryotic expression vector pCI to obtain the OR51S1 expression plasmid; the OR51S1 expression plasmid, receptor transport protein RTP1 and receptor expression enhancing protein REEP were transfected into HEK-293T cells using Lipofectamine2000 transfection reagent to express OR51S1 protein, and the plasmid transfection time was 24 hours.

[0048] Preparation of nanovesicles containing OR51S1 protein: 1.7 mg of L-homocysteine ​​sulfinic acid was dissolved in 10 mL of deionized water to obtain an L-homocysteine ​​sulfinic acid solution; 2.1 mg of 3-(3-acetylaminophenyl)propionic acid was dissolved in 10 mL of DMSO to obtain a 3-(3-acetylaminophenyl)propionic acid solution; 1 mg of cytochalasin B, 100 μL of L-homocysteine ​​sulfinic acid solution and 100 μL of 3-(3-acetylaminophenyl)propionic acid solution were added to 500 mL of serum-free DMEM medium. The HEK293T cells were incubated with the solution and mixed on a shaker at 37°C and 5% CO2 for 30 minutes. The mixture was centrifuged at 500×g for 10 minutes to remove the remaining cells, and the supernatant was centrifuged at 15000×g for 30 minutes to obtain the nanovesicle precipitate. The supernatant was discarded and the precipitate was resuspended in PBS containing 1% protease inhibitor cocktail. The total protein concentration was quantified using the Qubit protein assay kit and diluted to 100µg / mL with PBS containing 1% protease inhibitor cocktail. The nanovesicles were stored at -80°C.

[0049] Preparation of the sensor: Dissolve the nitrocellulose membrane in methanol and vortex for 10 minutes at room temperature to fully dissolve the nitrocellulose dispersion; add 4μL of the nitrocellulose dispersion to the surface of the working electrode, let it stand at room temperature until the methanol evaporates and dries, then add 15μL of 100µg / mL OR51S1 nanovesicle solution to the working electrode and let it stand at room temperature for 2 hours. The pore size of the nitrocellulose membrane is 0.45μm, and the area-to-volume ratio of the nitrocellulose membrane added to methanol is 1mm²:5μL; the working electrode area on the screen-printed gold electrode surface is 20mm 2 When the nitrocellulose dispersion is dropped onto the working electrode on the surface of the screen-printed gold electrode, it is required to cover only the working electrode and not contact the counter electrode and the reference electrode; when the nanovesicle solution is dropped, it is required to cover only the working electrode and not contact the counter electrode and the reference electrode.

[0050] Example 2: The difference between this example and Example 1 is only the preparation of nanovesicles containing OR51S1 protein.

[0051] Preparation of nanovesicles containing OR51S1 protein: 1.7 mg of L-homocysteine ​​sulfinic acid was dissolved in 10 mL of deionized water to obtain an L-homocysteine ​​sulfinic acid solution; 2.1 mg of 3-(3-acetylaminophenyl)propionic acid was dissolved in 10 mL of DMSO to obtain a 3-(3-acetylaminophenyl)propionic acid solution; 1 mg of cytochalasin B, 200 μL of L-homocysteine ​​sulfinic acid solution and 100 μL of 3-(3-acetylaminophenyl)propionic acid solution were added to 500 mL of serum-free DMEM culture medium, and HEK293T cells were incubated and mixed on a shaker at 37°C and 5% CO2 for 30 minutes. The mixture was centrifuged at 500×g for 10 minutes to remove the remaining cells, and the supernatant was centrifuged at 15000×g for 30 minutes to obtain a nanovesicle precipitate; the supernatant was discarded and the precipitate was resuspended with PBS containing 1% protease inhibitor cocktail. The total protein concentration was quantified using the Qubit protein assay kit, diluted to 100 µg / mL with PBS containing 1% protease inhibitor cocktail, and the nanovesicles were stored at −80°C.

[0052] Example 3: Compared with Example 1, the only difference between this example and Example 1 is the preparation of nanovesicles containing OR51S1 protein.

[0053] Preparation of nanovesicles containing OR51S1 protein: 1.7 mg of L-homocysteine ​​sulfinic acid was dissolved in 10 mL of deionized water to obtain an L-homocysteine ​​sulfinic acid solution; 2.1 mg of 3-(3-acetylaminophenyl)propionic acid was dissolved in 10 mL of DMSO to obtain a 3-(3-acetylaminophenyl)propionic acid solution; 50 mg of cocamidopropyl dimethylamine lactate was dissolved in 10 mL of deionized water to obtain a cocamidopropyl dimethylamine lactate solution; 1 mg of cell culture medium was added to 500 mL of serum-free DMEM medium. HEK293T cells were incubated with relaxin B, 100 μL of L-homocysteinesulfinic acid solution, 100 μL of 3-(3-acetamidophenyl) propionic acid solution, and 100 μL of cocamidopropyl dimethylamine lactate solution, and mixed on a shaker at 37°C and 5% CO2 for 30 min. The mixture was centrifuged at 500×g for 10 min to remove the remaining cells, and the supernatant was centrifuged at 15000×g for 30 min to obtain nanovesicle precipitation; the supernatant was discarded and the precipitate was resuspended in PBS containing 1% protease inhibitor cocktail. The total protein concentration was quantified using the Qubit protein assay kit, diluted to 100 μg / mL with PBS containing 1% protease inhibitor cocktail, and the nanovesicles were stored at -80°C.

[0054] Example 4: The difference between this example and Example 1 is only the preparation of nanovesicles containing OR51S1 protein.

[0055] Preparation of nanovesicles containing OR51S1 protein: 1.7 mg of L-homocysteine ​​sulfinic acid was dissolved in 10 mL of deionized water to obtain an L-homocysteine ​​sulfinic acid solution; 2.1 mg of 3-(3-acetylaminophenyl)propionic acid was dissolved in 10 mL of DMSO to obtain a 3-(3-acetylaminophenyl)propionic acid solution; 50 mg of cocamidopropyl dimethylamine lactate was dissolved in 10 mL of deionized water to obtain a cocamidopropyl dimethylamine lactate solution; 1 mg of cell culture medium was added to 500 mL of serum-free DMEM medium. HEK293T cells were incubated with relaxin B, 100 μL of L-homocysteinesulfinic acid solution, 100 μL of 3-(3-acetamidophenyl) propionic acid solution, and 200 μL of cocamidopropyl dimethylamine lactate solution. The mixture was mixed on a shaker at 37°C and 5% CO2 for 30 min. The mixture was centrifuged at 500 × g for 10 min to remove the remaining cells. The supernatant was centrifuged at 15000 × g for 30 min to obtain the nanovesicle precipitate. The supernatant was discarded and the precipitate was resuspended in PBS containing 1% protease inhibitor cocktail. The total protein concentration was quantified using the Qubit protein assay kit, diluted to 100 μg / mL with PBS containing 1% protease inhibitor cocktail, and the nanovesicles were stored at -80°C.

[0056] Comparative Example 1: The only difference between this comparative example and Example 1 is that L-homocysteine ​​sulfenic acid is not used in the preparation of nanovesicles containing OR51S1 protein.

[0057] Comparative Example 2: The only difference between this comparative example and Example 1 is that 3-(3-acetylaminophenyl)propionic acid is not used in the preparation of nanovesicles containing OR51S1 protein.

[0058] Comparative Example 3: The only difference between this comparative example and Example 1 is that L-homocysteinesulfinic acid and 3-(3-acetylaminophenyl)propionic acid are not used in the preparation of nanovesicles containing OR51S1 protein.

[0059] Experimental Example 1: Determination of the number of nanovesicles Test sample: undiluted nanovesicles containing OR51S1 protein prepared by the methods of various examples and comparative examples.

[0060] Test method: Nanovesicles containing OR51S1 protein were diluted to 10% with PBS containing 1% protease inhibitor cocktail. 7 -10 8 mL -1The concentration of nanovesicles was measured by the nanoparticle tracking analysis (NTA) technique. NTA tracks the Brownian motion of nanovesicles in the sample and combines Stokes' law to provide the concentration of nanovesicles (number of particles / mL) and calculate the number of vesicles.

[0061] The test results of the number of nanovesicles containing OR51S1 protein prepared by the present invention are as follows: Figure 1 As shown, the number of nanovesicles in Example 1 is 5.1×10 8 / mL, the number of nanovesicles in Example 2 is 5.0×10 8 / mL, the number of nanovesicles in Example 3 is 7.2×10 8 / mL, the number of nanovesicles in Example 4 is 7.1×10 8 / mL. From Example 1 to Example 3, it can be seen that in the presence of L-homocysteine ​​sulfinic acid and 3- (3-acetylaminophenyl) propionic acid, cells can release a certain number of nanovesicles according to normal physiological processes, and the dosage of each substance is 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 of nanovesicles or its stability. 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, so that the number of detectable nanovesicles increases. In Comparative Example 1, L-homocysteine ​​sulfinic acid was not used, and the number of vesicles was sharply reduced to 1.6×10 8 The number of vesicles was 2.0×10 / mL, which fully demonstrated the indispensability of L-homocysteine ​​sulfinic acid in the vesicle formation process. Its absence led to a significant inhibition of vesicle formation. In Comparative Example 2, 3-(3-acetylaminophenyl)propionic acid was not used, and the number of vesicles was 2.0×10 8 / mL, which is also significantly lower than that in Example 1, proving that 3-(3-acetylaminophenyl) propionic acid plays an important role in vesicle formation. In Comparative Example 3, L-homocysteinesulfinic acid and 3-(3-acetylaminophenyl) propionic acid were not used at the same time, and the number of vesicles was only 0.7×10 8 This indicates that these two substances together constitute the key basis for vesicle formation.

[0062] The number of nanovesicles containing OR51S1 protein prepared by the present invention is 5.0-7.0×10 8 In the range of pcs / mL, the number of nanovesicles was significantly enhanced, and the sensor's recognition and binding ability to geosmin was improved, thereby improving the sensitivity and accuracy of the sensor.

[0063] Experimental Example 2: Protein expression determination Test sample: undiluted nanovesicles containing OR51S1 protein prepared by the methods of various examples and comparative examples.

[0064] Test method: Nanovesicles containing OR51S1 protein were diluted with PBS containing 1% protease inhibitor cocktail, and the total protein concentration was quantified using the Qubit protein assay kit.

[0065] The protein expression test results of the nanovesicles containing OR51S1 protein prepared by the present invention are as follows: Figure 2 As shown, the protein concentration of Example 1 is 525µg / mL, the protein concentration of Example 2 is 564µg / mL, the protein concentration of Example 3 is 707µg / mL, and the protein concentration of Example 4 is 735µg / mL. From Example 1 to Example 2, it can be seen that the change in the amount of L-homocysteine ​​sulfinic acid mainly affects the total protein concentration, causing it to increase slightly, which indicates that the substance is involved in the process related to intracellular protein synthesis to a certain extent, and helps to increase the protein content in the vesicle. In Example 3, cocamidopropyl dimethylamine lactate was added on the basis of Example 1, and the total protein concentration increased significantly, which shows that the addition of cocamidopropyl dimethylamine lactate has a positive effect on the total protein concentration. It may promote the synthesis and stability of proteins by interacting with proteins in cells, so that more proteins can be effectively integrated into vesicles, and the total protein concentration is increased. In Example 4, the concentration of cocamidopropyl dimethylamine lactate was increased to 10µg / mL, and the total protein concentration reached the highest, reflecting the positive effect of cocamidopropyl dimethylamine lactate on protein synthesis. In comparative example 1, L-homocysteine ​​sulfinic acid was not used, and the total protein concentration was only 380mg / mL, which embodies the key role of L-homocysteine ​​sulfinic acid in intracellular physiological processes, and its absence causes a significant reduction in total protein concentration, and plays a vital role in promoting the protein synthesis process. Comparative example 2 does not use 3-(3-acetylaminophenyl) propionic acid, and the total protein concentration is 423mg / mL, proving that 3-(3-acetylaminophenyl) propionic acid is of great significance to protein synthesis and vesicle formation, and its absence can significantly reduce the total protein concentration, and the number of vesicles is also significantly reduced. Comparative example 3 does not use L-homocysteine ​​sulfinic acid and 3-(3-acetylaminophenyl) propionic acid at the same time, and the total protein concentration is as low as 356mg / mL. The common absence of these two substances makes the total protein concentration extremely low, further emphasizing that they jointly constitute the key basis of intracellular protein synthesis, which is indispensable. The total protein concentration of the nanovesicles containing OR51S1 protein prepared by the present invention is in the range of 525-735 µg / mL. This relatively stable and high level of total protein concentration significantly enhances the performance of the nanovesicles, greatly improves the sensor's recognition and binding ability to geosmin, thereby effectively improving the sensitivity and accuracy of the sensor.

[0066] The schematic diagram of the cyclic voltammetry curve of geosmin-OR51S1 is as follows Figure 3 As shown, the biosensor for detecting geosmin based on human olfactory receptor OR51S1 prepared in Example 1 is used to detect geosmin in an aqueous solution, comprising the following steps: using deionized water to prepare a redox couple solution containing 5 mmol / L potassium ferrocyanide, 5 mmol / L potassium ferrocyanide, and 0.1 mol / L potassium chloride, using deionized water to dilute the geosmin methanol standard solution 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, and setting 0 ng / L was used as a blank control; the test solution was mixed with an equal volume of a redox couple solution to obtain a mixed test solution; the working electrode, counter electrode and reference electrode of the OR51S1 nanovesicle-modified microelectrode sensor were connected to the electrochemical workstation one by one through adapters and wires, 200 μL of the mixed test solution was dropped on the surface of the microelectrode to ensure that the three electrodes were completely immersed, and the reduction peak current was measured by cyclic voltammetry, and the parameters of the cyclic voltammetry were set as follows: scan speed 50 mV / s, scan range -0.3 V to 0.7 V, and scan number 2-3 circles.

[0067] The embodiments and / or implementation methods described above are only used to illustrate the preferred embodiments and / or implementation methods for realizing the technology of the present invention, and are not intended to limit the implementation methods of the technology of the present invention in any form. Any technical personnel in this field may make slight changes or modifications to other equivalent embodiments without departing from the scope of the technical means disclosed in the content of the present invention, but they should still be regarded as technologies or embodiments that are essentially the same as the present invention.

[0068] This article uses specific examples to illustrate the principles and implementation methods of this application. The description of the above embodiments is only used to help understand the method and its core ideas of this application. The above is only the preferred implementation method of this application. It should be pointed out that due to the limitations of textual expression and the objective existence of infinite specific structures, ordinary technicians in this technical field can make several improvements, modifications or changes without departing from the principles of this application, and can also combine the above technical features in an appropriate manner; these improvements, modifications, changes or combinations, or the direct application of the concept and technical solution of the invention to other occasions without improvement, should be regarded as the scope of protection of this 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; The nanovesicles containing OR51S1 protein were added to the working electrode of the screen-printed gold electrode, which was modified with a nitrocellulose layer, and the nanovesicles containing OR51S1 protein were located in the nitrocellulose layer.

2. The method for preparing a biosensor for detecting geosmin based on human olfactory receptor OR51S1 according to claim 1, characterized in that: The cell culture fluid uses serum-free DMEM culture medium as a diluent.

3. The method for preparing a biosensor for detecting geosmin based on human olfactory receptor OR51S1 according to claim 2, characterized in that: The mass volume ratio of the L-homocysteinesulfinic acid to the serum-free DMEM medium is 3.4-34 μg:100-1000 mL; or the mass volume ratio of the 3-(3-acetylaminophenyl)propionic acid to the serum-free DMEM medium is 4.2-42 μg:100-1000 mL.

4. The method for preparing a biosensor for detecting geosmin based on human olfactory receptor OR51S1 according to claim 1, characterized in that: The cell culture medium also contains cytochalasin B.

5. The method for preparing a biosensor for detecting geosmin based on human olfactory receptor OR51S1 according to claim 4, characterized in that: The mass volume ratio of the cytochalasin B to the serum-free DMEM culture medium is 1-20 mg: 100-1000 mL.

6. 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 .

7. 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.

8. The method for preparing a biosensor for detecting geosmin based on human olfactory receptor OR51S1 according to claim 7, 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.

9. The method for preparing a biosensor for detecting geosmin based on human olfactory receptor OR51S1 according to claim 7, 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.

10. A biosensor for detecting geosmin based on human olfactory receptor OR51S1 prepared by the method according to any one of claims 1 to 9.

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