Preparation method of biosensor for detecting 2-methylisoborneol

Abstract

The invention discloses a preparation method of a biosensor for detecting 2-methylisoborneol, and belongs to the technical field of biosensors. In particular to a preparation method of an olfactory receptor heterologous expression system, a preparation method of an olfactory receptor cell nano-vesicle, a preparation method of a 2-hydroxyethyl ferrocenecarboxylate solution, a preparation method of an oxidized cellulose dispersion liquid, a preparation method of a polyvinyl alcohol solution and a preparation method of a biosensor. The biosensor prepared by the method is high in electric signal intensity and high in sensitivity.

Description

Technical Field

[0001] The invention belongs to the technical field of biosensors, and particularly relates to a method for preparing a biosensor for detecting 2-methylisoborneol. Background Art

[0002] 2-Methylisoborneol is a metabolite produced by blue-green algae and other microorganisms. It is a key earthy smell substance in drinking water. It has a strong earthy smell and can be sensed by human sense of smell. The presence of 2-Methylisoborneol seriously affects the quality of drinking water and is related to human health and safety.

[0003] The content of 2-methylisoborneol in drinking water is usually very low, at the level of nanograms per liter. At present, it is usually determined by gas chromatography-mass spectrometry (GC-MS) or triple quadrupole gas chromatography tandem mass spectrometry (GC-MS / MS) combined with pretreatment technologies such as liquid-liquid extraction and solid phase microextraction. The pretreatment of traditional analytical methods is relatively complex and the detection instruments are large, which makes it difficult to meet the needs of on-site detection, especially the ability to respond to sudden odor events is seriously insufficient. The electrochemical method can convert the concentration signal into an easily measurable electrical signal to realize the detection of 2-methylisoborneol. In addition, the operation of electrochemical sensors is relatively simple, does not require complex sample pretreatment steps, and is easy to deploy and use quickly on site. However, 2-methylisoborneol itself lacks electrochemical activity, and it is difficult to react directly on the electrode surface, and it is difficult to reach the extremely low detection threshold of 2-methylisoborneol, which limits the application of electrochemical methods.

[0004] In view of the characteristic odor of 2-methylisoborneol, the biosensor can specifically identify the target molecule, thereby improving the selectivity of detection. In some previous bionic sensor studies, olfactory tissues or cells were used as sensitive elements, which had defects such as harsh living conditions, many intracellular interference reactions and limited sensor size; odor binding proteins lack specificity and have weak binding ability to odor molecules. Therefore, there is an urgent need for a rapid, sensitive and cost-effective means of detecting 2-methylisoborneol in drinking water to improve the efficiency and accuracy of water quality monitoring. Summary of the invention

[0005] The object of the present invention is to provide a method for preparing a biosensor for detecting 2-methylisoborneol, wherein the surface of a working electrode is modified with nanovesicles containing olfactory receptor proteins.

[0006] The technical solution adopted by the present invention to achieve the above-mentioned purpose is: A biosensor for detecting 2-methylisoborneol comprises: a screen-printed gold electrode, and a carrier film layer modified on the working electrode surface of the screen-printed gold electrode, wherein the carrier film layer contains nanovesicles, and the nanovesicles contain olfactory receptor proteins, wherein the olfactory receptor proteins are OR3A4 proteins; and the carrier film layer comprises nitrocellulose, oxidized cellulose, and 2-hydroxyethyl ferrocenylcarboxylate. Nitrocellulose, oxidized cellulose, and 2-hydroxyethyl ferrocenylcarboxylate form a stable membrane structure together, so that the nanovesicles can be evenly distributed therein, and the 2-methylisoborneol molecules can diffuse to the nanovesicle region where the OR3A4 protein is located at an appropriate speed, while promoting electron transfer, realizing the generation and amplification of signals in the electrochemical biosensor, and improving the accuracy and sensitivity of detection.

[0007] The invention provides a method for preparing a biosensor for detecting 2-methylisoborneol. The method comprises the following steps: mixing a nitrocellulose dispersion, an oxidized cellulose dispersion and a 2-hydroxyethyl ferrocenecarboxylate solution, dripping the mixture onto a working electrode surface of a screen-printed gold electrode, and then dripping a nanovesicle solution. The mixture is allowed to stand and dry to obtain a biosensor for detecting 2-methylisoborneol.

[0008] Preferably, the nitrocellulose is measured based on the area of ​​the nitrocellulose membrane, and the area mass ratio of the nitrocellulose, oxidized cellulose and 2-hydroxyethyl ferrocene carboxylate in the carrier membrane layer is 2000-2500 mm 2 :0.05-0.2g:1-5g.

[0009] Preferably, the mass area ratio of total protein to nitrocellulose in the nanovesicles is 3.75-5 mg: 2000-2500 mm 2 .

[0010] 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.

[0011] Preferably, the oxidized cellulose dispersion is obtained by dispersing oxidized cellulose in deionized water, and the mass volume ratio of oxidized cellulose to deionized water is 0.5-2g:10-100mL.

[0012] Preferably, the 2-hydroxyethyl ferrocenylcarboxylate solution is obtained by dissolving 2-hydroxyethyl ferrocenylcarboxylate in methanol, and the mass volume ratio of 2-hydroxyethyl ferrocenylcarboxylate to methanol is 1-5 g:10-100 mL.

[0013] Preferably, the volume ratio of the nitrocellulose dispersion, the oxidized cellulose dispersion and the 2-hydroxyethyl ferrocenecarboxylate solution is 1-10:1-10:1-10.

[0014] Preferably, the volume ratio of the nitrocellulose dispersion to the nanovesicle solution is 2-10:7.5-37.5.

[0015] 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 .

[0016] More preferably, polyvinyl alcohol can be added in the preparation of the carrier membrane layer in the biosensor for detecting 2-methylisoborneol, and the area mass ratio of nitrocellulose, polyvinyl alcohol, oxidized cellulose and 2-hydroxyethyl ferrocene formate solution is 2000-2500 mm 2 :0.05-0.2g:0.05-0.2g:1-5g. With the addition of polyvinyl alcohol, under the joint action of the carrier film layer, the nanovesicles can be evenly distributed on the surface of the working electrode and maintain a relatively fixed position, so that the biosensor can better adapt to the environment in the body, reduce the adverse effects on cells, and improve the biocompatibility and stability of the biosensor.

[0017] The present invention also provides a method for preparing olfactory receptor cell nanovesicles, comprising: OR3A4 was selected as the human olfactory receptor of 2-methylisoborneol. According to the gene sequence information in the Olfactory Receptor Database, the OR3A4 gene was chemically synthesized and cloned into the restriction sites MluI and NotI of the mammalian eukaryotic expression vector pCI. Two fusion expression tags, Flag-tag and the first 20 amino acids of human rhodopsin (Rho-tag), were inserted between the restriction sites NheI and EcoRI of the pCI vector to obtain the olfactory receptor expression plasmid. HEK-293T cells with good growth status were selected, and plasmids were introduced into cells by liposome transfection to express olfactory receptor protein. 12-36 hours after plasmid transfection, HEK293T cells were cultured in serum-free DMEM medium containing cytochalasin B at 36-37°C and 4-5% CO 2 Incubate for 20-40 minutes under the same conditions, centrifuge the mixture to remove the remaining cells, centrifuge the supernatant to obtain the nanovesicle precipitate, resuspend it in PBS containing a protease inhibitor cocktail, and obtain the olfactory receptor cell nanovesicle. Use the Qubit instrument to quantify the olfactory receptor protein concentration in the nanovesicle, and dilute it to a working concentration of 10-100µg / mL with PBS containing a protease inhibitor cocktail.

[0018] Preferably, the mass volume ratio of cytochalasin B to serum-free DMEM medium is 1-10 mg: 100-500 mL.

[0019] Preferably, the volume ratio of the protease inhibitor cocktail to PBS in the PBS containing the protease inhibitor cocktail is 1-2 mL:100-200 mL.

[0020] Preferably, the mixed solution is centrifuged at a speed of 300-700×g for 5-15 min; the supernatant is centrifuged at a speed of 13000-17000×g for 20-40 min.

[0021] The present invention also provides a method for preparing 2-hydroxyethyl ferrocenecarboxylate, comprising: Dissolve ferrocenecarboxylic acid in carbon tetrachloride, stir and dissolve under nitrogen protection, continue to pass nitrogen for 20-40 minutes, seal the reaction system and add oxalyl chloride, stir and react at room temperature for 6-10 hours, and remove the carbon tetrachloride solvent and unreacted oxalyl chloride by rotary evaporation to obtain ferrocenecarboxylic acid chloride; Ethylene glycol and dichloromethane were mixed and dissolved, triethylamine was added, and the mixture was placed in an ice-water mixed bath under nitrogen protection and stirred for 15-45 minutes. Ferrocenecarbonyl chloride was dissolved in dichloromethane and added to the reaction system, and the ice-water bath was removed. The mixture was stirred at room temperature for 12-36 hours, and the mixture was washed with saturated brine, and the remaining solvent was removed by rotary evaporation to obtain 2-hydroxyethyl ferrocenecarboxylate.

[0022] Preferably, the mass volume ratio of ferrocenecarboxylic acid to carbon tetrachloride is 1-5g:20-100mL.

[0023] Preferably, the mass volume ratio of ferrocenecarboxylic acid to oxalyl chloride is 1-5 g:1-5 mL.

[0024] Preferably, the mass volume ratio of ethylene glycol to dichloromethane is 1-5g:10-50mL.

[0025] Preferably, the mass volume ratio of ethylene glycol to triethylamine is 1-5g:0.5-2.5mL.

[0026] Preferably, the mass ratio of ethylene glycol to ferrocenecarbonyl chloride is 1-5:4-20.

[0027] Preferably, the mass volume ratio of ferrocenecarbonyl chloride to dichloromethane is 4-20 g:10-50 mL.

[0028] The present invention also provides a method for preparing oxidized cellulose slurry, comprising: The microcrystalline cellulose is dispersed in a solution containing 2,2,6,6-tetramethylpiperidin-1-oxyl free radical (TEMPO) and sodium bromide, sodium hypochlorite is added, the pH is adjusted to 9.8-10.2, the solution is heated to 35-45°C and soaked for 2-4 hours, and the TEMPO-oxidized cellulose is washed with deionized water; the TEMPO-oxidized cellulose is re-dispersed in deionized water, and treated with an ultrasonic cell crusher to obtain an oxidized cellulose slurry.

[0029] Preferably, the mass ratio of microcrystalline cellulose, TEMPO and sodium bromide is 1-10:0.016-0.16:0.1-1.

[0030] Preferably, the mass ratio of microcrystalline cellulose to sodium hypochlorite is 1-10:0.74-7.4.

[0031] Preferably, the mass volume ratio of TEMPO-oxidized cellulose to deionized water is 1-10 g:10-100 mL.

[0032] Preferably, the power of the ultrasonic cell pulverizer is 300 W, and the treatment time is 10-20 min.

[0033] The present invention also provides a method for preparing a biosensor, comprising: The cellulose nitrate membrane is dispersed in methanol to obtain a cellulose nitrate dispersion; 2-hydroxyethyl ferrocene carboxylate is dissolved in methanol to obtain a 2-hydroxyethyl ferrocene carboxylate solution; the oxidized cellulose slurry is dispersed in deionized water, heated at 75-85°C and stirred until completely dispersed to form a uniform solution; the cellulose nitrate dispersion, the oxidized cellulose dispersion and the hydroxyethyl ferrocene carboxylate solution are mixed, the pH value is adjusted to 7.0-7.2, and stirred at room temperature to obtain a mixed solution; the mixed solution is dripped onto the working electrode on the surface of the screen-printed gold electrode, and allowed to stand at room temperature until the methanol and water evaporate and dry; a nanovesicle solution containing olfactory receptor proteins is dripped onto the surface of the gold electrode; the modified microelectrode is allowed to stand at room temperature for 1-3 hours to obtain a biosensor.

[0034] 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.

[0035] Preferably, the mass volume ratio of 2-hydroxyethyl ferrocenecarboxylate to methanol is 1-5 g:10-100 mL.

[0036] Preferably, the mass volume ratio of the oxidized cellulose slurry to deionized water is 0.5-2 g:10-100 mL.

[0037] Preferably, the volume ratio of the nitrocellulose dispersion, the oxidized cellulose dispersion and the 2-hydroxyethyl ferrocenecarboxylate solution is 1-10:1-10:1-10.

[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 olfactory receptor proteins with specific recognition ability for 2-methylisoborneol. The biosensor is modified with 2-hydroxyethyl ferrocene carboxylate, oxidized cellulose and polyvinyl alcohol, thereby having the following beneficial effects: the carrier film layer provides a stable fixed environment for the nanovesicles containing the olfactory receptors and promotes the transfer of electrons, and the biocompatibility and stability of the biosensor are increased. Therefore, the present invention is a biosensor with a strong electrical signal and a preparation method thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 is a schematic diagram of the structure of the biosensor; Figure 2 This is a graph showing the test results of the vesicle shedding rate of the biosensor; Figure 3 This is a graph showing the electrical signal strength test results of the biosensor. DETAILED DESCRIPTION

[0043] 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.

[0044] 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.

[0045] Embodiment 1: A method for preparing a biosensor for detecting 2-methylisoborneol, comprising: Construction of olfactory receptor heterologous expression system: OR3A4 was selected as the human olfactory receptor for 2-methylisoborneol, and HEK-293T cells were used as host cells. According to the gene sequence information in the Olfactory Receptor Database, the OR3A4 gene was chemically synthesized and cloned into the restriction sites MluI and NotI of the mammalian eukaryotic expression vector pCI. Two fusion expression tags, Flag-tag and the first 20 amino acids of human rhodopsin (Rho-tag), were inserted between the restriction sites NheI and EcoRI of the pCI vector to obtain the olfactory receptor expression plasmid. HEK-293T cells with good growth status were selected, and the plasmid was introduced into the cells by liposome transfection to express the olfactory receptor protein.

[0046] Preparation of olfactory receptor cell nanovesicles: 24 h after plasmid transfection, HEK293T cells were cultured in serum-free DMEM medium containing 20 µg / mL cytochalasin B at 37°C and 5% CO. 2 The mixture was incubated for 30 min under the same conditions, and the mixture was centrifuged at 500 × g for 10 min. The supernatant was centrifuged at 15,000 × g for 30 min to obtain nanovesicle precipitates, which were resuspended in PBS containing a protease inhibitor cocktail to obtain olfactory receptor cell nanovesicles. The olfactory receptor protein concentration in the nanovesicles was quantitatively determined using a Qubit instrument and diluted to a working concentration of 100 µg / mL.

[0047] Preparation of 2-hydroxyethyl ferrocenylcarboxylate: Under nitrogen protection, 1.15g of ferrocenylcarboxylic acid was mixed with 25mL of carbon tetrachloride and stirred until the ferrocenylcarboxylic acid was completely dissolved, and the system was sealed after nitrogen was continuously passed for 30min. 1.2mL of oxalyl chloride was added, and the carbon tetrachloride solvent and unreacted oxalyl chloride were removed by rotary evaporation to obtain ferrocenylcarboxylate; 3.1g of ethylene glycol and 25mL of dry dichloromethane were mixed and stirred, 1.4mL of triethylamine was added, and the mixture was placed in an ice-water mixed bath and stirred for 30min under nitrogen protection; 12g of the above-prepared ferrocenylcarboxylate was dissolved in 20mL of dichloromethane and added to the reaction system, the ice-water bath was removed, and the mixture was stirred at room temperature for 24h, then washed with saturated brine, and the remaining solvent was removed by rotary evaporation to obtain 2-hydroxyethyl ferrocenylcarboxylate.

[0048] Preparation of oxidized cellulose slurry: disperse 1 g of microcrystalline cellulose in a solution of 0.016 g of 2,2,6,6-tetramethylpiperidin-1-oxyl free radical (TEMPO) and 0.1 g of sodium bromide, add 0.74 g of sodium hypochlorite, adjust the pH to 10.0, heat to 40°C and soak for 3 h, then wash the TEMPO-oxidized cellulose with deionized water; redisperse 1 g of TEMPO-oxidized cellulose in 10 mL of deionized water, treat with an ultrasonic cell crusher for 15 min to obtain oxidized cellulose slurry, and store it in a frozen and sealed container.

[0049] Preparation of biosensor: Disperse nitrocellulose membrane in methanol to obtain nitrocellulose dispersion; dissolve 2.3g of 2-hydroxyethyl ferrocene formate in 10mL of methanol to obtain 2-hydroxyethyl ferrocene formate solution; disperse 1g of oxidized cellulose in 100mL of deionized water, heat to 80°C, and stir continuously to form oxidized cellulose dispersion; mix 1mL of nitrocellulose dispersion, 1mL of oxidized cellulose dispersion and 1mL of 2-hydroxyethyl ferrocene formate solution, adjust the pH value to 7.0, and stir at room temperature to obtain a mixed solution; drop 12μL of the mixed solution on the working electrode surface of the screen-printed gold electrode surface, and let stand at room temperature until methanol and water evaporate and dry; drop 15μL of the nanovesicle solution containing olfactory receptor protein on the working electrode surface of the screen-printed gold electrode surface, and let stand at room temperature for 2h to obtain a biosensor. The pore size of the nitrocellulose membrane is 0.45μm, and the area-to-volume ratio of the nitrocellulose membrane dispersed in methanol is 1mm 2 :5μL; the working electrode area on the screen-printed gold electrode surface is 20mm 2 When the mixed solution 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] Embodiment 2: A method for preparing a biosensor for detecting 2-methylisoborneol, The difference between this embodiment and embodiment 1 is only the preparation of the biosensor.

[0051] Preparation of biosensor: Disperse nitrocellulose membrane in methanol to obtain nitrocellulose dispersion; dissolve 2.3g of 2-hydroxyethyl ferrocene formate in 10mL of methanol to obtain 2-hydroxyethyl ferrocene formate solution; disperse 2g of oxidized cellulose in 100mL of deionized water, heat to 80°C, and stir continuously to form oxidized cellulose dispersion; mix 1mL of nitrocellulose dispersion, 1mL of oxidized cellulose dispersion and 1mL of 2-hydroxyethyl ferrocene formate solution, adjust the pH value to 7.0, and stir at room temperature to obtain a mixed solution; drop 12μL of the mixed solution on the working electrode surface of the screen-printed gold electrode surface, and let stand at room temperature until methanol and water evaporate and dry; drop 15μL of the nanovesicle solution containing olfactory receptor protein on the gold electrode surface, and let stand at room temperature for 2h to obtain a biosensor. The pore size of the nitrocellulose membrane is 0.45μm, and the area-to-volume ratio of the nitrocellulose membrane dispersed in methanol is 1mm 2 :5μL; the working electrode area on the screen-printed gold electrode surface is 20mm 2 When the mixed solution 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.

[0052] Embodiment 3: A method for preparing a biosensor for detecting 2-methylisoborneol, The difference between this embodiment and embodiment 1 is only the preparation of the biosensor.

[0053] Preparation of biosensor: Disperse nitrocellulose membrane in methanol to obtain nitrocellulose dispersion; dissolve 2.3g of 2-hydroxyethyl ferrocenecarboxylate in 10mL of methanol to obtain 2-hydroxyethyl ferrocenecarboxylate solution; disperse 1g of oxidized cellulose in 100mL of deionized water, heat to 80°C, and continue stirring to form an oxidized cellulose dispersion; dissolve 1g of polyvinyl alcohol in 100mL of deionized water, heat to 80°C and stir until completely dissolved to obtain a polyvinyl alcohol solution; mix 1mL of polyvinyl alcohol solution, 1mL of nitrocellulose dispersion, 1mL of oxidized cellulose dispersion and 1mL of 2-hydroxyethyl ferrocenecarboxylate solution, adjust the pH value to 7.0, and stir at room temperature to obtain a mixed solution; drop 16μL of the mixed solution on the working electrode surface of the screen-printed gold electrode surface, and let it stand at room temperature until methanol and water evaporate and dry; drop 15μL of the nanovesicle solution containing olfactory receptor protein on the gold electrode surface, and let it stand at room temperature for 2h to obtain a biosensor. The pore size of the nitrocellulose membrane is 0.45 μm, and the area-to-volume ratio of the nitrocellulose membrane dispersed in methanol is 1 mm 2 :5μL; the working electrode area on the screen-printed gold electrode surface is 20mm2 When the mixed solution 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.

[0054] Embodiment 4: A method for preparing a biosensor for detecting 2-methylisoborneol, The difference between this embodiment and embodiment 1 is only the preparation of the biosensor.

[0055] Preparation of biosensor: Disperse nitrocellulose membrane in methanol to obtain nitrocellulose dispersion; dissolve 2.3g of 2-hydroxyethyl ferrocenecarboxylate in 10mL of methanol to obtain 2-hydroxyethyl ferrocenecarboxylate solution; disperse 1g of oxidized cellulose in 100mL of deionized water, heat to 80°C, and continue stirring to form an oxidized cellulose dispersion; dissolve 2g of polyvinyl alcohol in 100mL of deionized water, heat to 80°C and stir until completely dissolved to obtain a polyvinyl alcohol solution; mix 1mL of polyvinyl alcohol solution, 1mL of nitrocellulose dispersion, 1mL of oxidized cellulose dispersion and 1mL of 2-hydroxyethyl ferrocenecarboxylate solution, adjust the pH value to 7.0, and stir at room temperature to obtain a mixed solution; drop 16μL of the mixed solution on the working electrode surface of the screen-printed gold electrode surface, and let it stand at room temperature until methanol and water evaporate and dry; drop 15μL of the nanovesicle solution containing olfactory receptor protein on the gold electrode surface, and let it stand at room temperature for 2h to obtain a biosensor. The pore size of the nitrocellulose membrane is 0.45 μm, and the area-to-volume ratio of the nitrocellulose membrane dispersed in methanol is 1 mm 2 :5μL; the working electrode area on the screen-printed gold electrode surface is 20mm 2 When the mixed solution 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.

[0056] Comparative Example 1: The difference between this comparative example and Example 1 is only the preparation of the biosensor.

[0057] Preparation of biosensor: Disperse nitrocellulose membrane in methanol to obtain nitrocellulose dispersion; dissolve 2.3g of 2-hydroxyethyl ferrocene carboxylate in 10mL of methanol to obtain 2-hydroxyethyl ferrocene carboxylate solution; mix 1mL of nitrocellulose dispersion with 1mL of 2-hydroxyethyl ferrocene carboxylate solution, adjust the pH value to 7.0, and stir at room temperature to obtain a mixed solution; drip 8μL of the mixed solution on the working electrode surface of the screen-printed gold electrode surface, and let it stand at room temperature until methanol and water evaporate and dry; drip 15μL of the nanovesicle solution containing olfactory receptor protein on the working electrode surface of the screen-printed gold electrode surface, and let it stand at room temperature for 2h to obtain a biosensor. The pore size of the nitrocellulose membrane is 0.45μm, and the area-to-volume ratio of the nitrocellulose membrane dispersed in methanol is 1mm 2 :5μL; the working electrode area on the screen-printed gold electrode surface is 20mm 2 When the mixed solution 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.

[0058] Comparative Example 2: The difference between this comparative example and Example 1 is only the preparation of the biosensor.

[0059] Preparation of biosensor: Disperse nitrocellulose membrane in methanol to obtain nitrocellulose dispersion; disperse 1g oxidized cellulose in 100mL deionized water, heat to 80°C, and stir continuously to form oxidized cellulose dispersion; mix 1mL nitrocellulose dispersion with 1mL oxidized cellulose dispersion, adjust pH to 7.0, and stir at room temperature to obtain a mixed solution; drip 8μL of the mixed solution onto the working electrode surface of the screen-printed gold electrode, and let stand at room temperature until methanol and water evaporate and dry; drip 15μL of the nanovesicle solution containing olfactory receptor protein onto the working electrode surface of the screen-printed gold electrode, and let stand at room temperature for 2h to obtain a biosensor. The pore size of the nitrocellulose membrane is 0.45μm, and the area-to-volume ratio of the nitrocellulose membrane dispersed in methanol is 1mm 2 :5μL; the working electrode area on the screen-printed gold electrode surface is 20mm 2 When the mixed solution 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.

[0060] Comparative Example 3: The difference between this comparative example and Example 1 is only the preparation of the biosensor.

[0061] Preparation of biosensor: Disperse nitrocellulose membrane in methanol to obtain nitrocellulose dispersion; drip 4 μL of nitrocellulose dispersion onto the working electrode surface of the screen-printed gold electrode surface, and let stand at room temperature until methanol and water evaporate and dry; drip 15 μL of nanovesicle solution containing olfactory receptor protein onto the working electrode surface of the screen-printed gold electrode surface, and let stand at room temperature for 2 hours to obtain the biosensor. The pore size of the nitrocellulose membrane is 0.45 μm, and the area-to-volume ratio of the nitrocellulose membrane dispersed in methanol is 1 mm 2 :5μL; the working electrode area on the screen-printed gold electrode surface is 20mm 2 When the mixed solution 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.

[0062] The schematic diagram of the structure of the biosensor prepared in Example 1 of the present invention is as follows Figure 1 As shown, the working electrode is a screen-printed gold electrode modified with nanovesicles containing olfactory receptor proteins, the carrier membrane layer includes 2-hydroxyethyl ferrocenecarboxylate, oxidized cellulose, polyvinyl alcohol and nitrocellulose, the counter electrode is a screen-printed gold electrode, and the reference electrode is silver / silver chloride.

[0063] Test Example 1: Vesicle Shedding Rate Test sample: biosensor prepared by the methods of various embodiments and comparative examples.

[0064] Test method: The concentration of olfactory receptor protein in the nanovesicles initially adsorbed on the biosensor surface was determined using a Qubit instrument (C 0 The biosensor was placed in drinking water at room temperature and shaken for 24 hours. The concentration of olfactory receptor protein in the remaining nanovesicles on the surface of the biosensor was measured again using the Qubit instrument (C 1 ). According to the formula: Vesicle shedding rate = (C 0 - C 1 ) / C 0 × 100% to calculate the vesicle shedding rate.

[0065] The test results of the vesicle shedding rate prepared by the present invention are as follows Figure 2As shown, the vesicle shedding rate of Example 1 is 11.7%, the vesicle shedding rate of Example 2 is 13.2%, the vesicle shedding rate of Example 3 is the lowest, which is 7.8%, and the vesicle shedding rate of Example 4 is 10.3%. It can be seen from Example 1 to Example 3 that the optimization of the preparation process, especially after the addition of polyvinyl alcohol, reduces the vesicle shedding rate, indicating that polyvinyl alcohol helps to enhance the binding force between the vesicle and the electrode surface. Compared with Example 1, Example 2 and Example 4 compared with Example 3, the amount and ratio of each component were changed during the preparation of the biosensor, so that the vesicle shedding rate increased, indicating that the appropriate amount of solution has a positive effect on the stability of the vesicle. In the case of Comparative Example 1 without the use of oxidized cellulose dispersion, the vesicle shedding rate reached 24.3%, which is significantly higher in comparison, indicating that oxidized cellulose plays an important role in the stable fixation of the vesicle. Comparative Example 2 did not use 2-hydroxyethyl ferrocene formate solution, and its vesicle shedding rate was 23.0%, which was also significantly higher than Example 3, indicating that 2-hydroxyethyl ferrocene formate helps to enhance the binding stability of the vesicle and the electrode. Comparative Example 3 uses neither 2-hydroxyethyl ferrocene formate nor oxidized cellulose dispersion, and its vesicle shedding rate is as high as 40.8%, further proving the key role of these two substances in reducing the vesicle shedding rate. Compared with Comparative Examples 1-3, Experimental Example 3 fully demonstrates that after the nanovesicles are modified with 2-hydroxyethyl ferrocene formate, oxidized cellulose and polyvinyl alcohol according to the method of the present invention, the vesicle shedding rate is significantly reduced. The overall vesicle shedding rate of the biosensor prepared by the present invention is in the range of 7-14%, which significantly improves the stability of the biosensor.

[0066] Test Example 2: Electrical Signal Strength Test sample: biosensor prepared by the methods of various embodiments and comparative examples.

[0067] Test method: Ultrapure water was used to prepare an electrolyte solution containing 5 mmol / L potassium ferrocyanide, 5 mmol / L potassium ferrocyanide and 0.1 mol / L potassium chloride. The microelectrode modified with nanovesicles was connected to the electrochemical workstation, and 100 μL of electrolyte solution was dripped on the surface of the printed electrode to ensure that the working electrode, reference electrode and counter electrode were completely immersed, and cyclic voltammetry was performed. The parameters were set as a scan speed of 0.05 V / s, a scan range of 0.7 V to -0.3 V, and a scan number of 2 circles, and the peak current signal intensity of different biosensors was measured.

[0068] The present invention uses potassium ferrocyanide-potassium ferrocyanide redox system to characterize the electrochemical detection effect of the biosensor. The peak current signal intensity test results are as follows: Figure 3As shown, the oxidation peak current potential of Example 1 is 6.7μA, the oxidation peak current potential of Example 2 is 6.8μA, the oxidation peak current potential of Example 3 is the highest, reaching 9.0μA, and the current of Example 4 is 8.5μA. From Example 1 to Example 3, the electrical signal intensity gradually increases, which is closely related to the changes in the amount and ratio of the oxidized cellulose dispersion, 2-hydroxyethyl ferrocene formate solution and polyvinyl alcohol used in the preparation of the biosensor in each embodiment. After optimizing the solution ratio and adding polyvinyl alcohol in Example 3, the current reached the maximum value of 9.0μA, indicating that polyvinyl alcohol and other components synergistically improved the electrical signal response intensity of the biosensor. Example 4 changes the solution dosage on the basis of Example 3, and the electrical signal intensity slightly decreases to 8.5μA, which once again reflects the important influence of the dosage and ratio of each component on the performance of the biosensor. Comparative Example 1 does not use an oxidized cellulose dispersion, and its current is only 4.1μA, which is much lower than Example 3, indicating that oxidized cellulose has a positive effect on enhancing electrical signals. Comparative Example 2 did not use 2-hydroxyethyl ferrocene formate solution, and the current was 3.9μA, which was also significantly lower than Example 3, indicating that 2-hydroxyethyl ferrocene formate plays an important role in improving the strength of electrical signals. Comparative Example 3 used neither 2-hydroxyethyl ferrocene formate nor oxidized cellulose dispersion, and the lowest current was only 3.2μA, further confirming the key role of these two substances in improving the strength of the electrical signal of the biosensor. Compared with Comparative Examples 1-3, Experimental Example 3 shows that after using 2-hydroxyethyl ferrocene formate, oxidized cellulose and polyvinyl alcohol to modify nanovesicles according to the method of the present invention, the peak current signal intensity of the biosensor has been significantly increased. The peak current signal intensity of the biosensor prepared by the present invention is the highest, and the current is between 6-9μA, which significantly improves the response intensity of the biosensor to detect electrical signals.

[0069] 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.

[0070] 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 biosensor for detecting 2-methylisoborneol, comprising: Screen-printed gold electrodes, and modified carrier membrane layers on the working electrode surfaces of the screen-printed gold electrodes, wherein the carrier membrane layers contain nanovesicles, and the nanovesicles contain olfactory receptor proteins, wherein the olfactory receptor proteins are OR3A4 proteins; The carrier film layer comprises nitrocellulose, oxidized cellulose and 2-hydroxyethyl ferrocenecarboxylate.

2. A biosensor for detecting 2-methylisoborneol according to claim 1, characterized in that: The nitrocellulose is measured based on the area of ​​the nitrocellulose membrane, and the area mass ratio of the nitrocellulose, oxidized cellulose and 2-hydroxyethyl ferrocene carboxylate in the carrier membrane layer is 2000-2500 mm 2 :0.05-0.2g:1-5g.

3. A biosensor for detecting 2-methylisoborneol according to claim 1, characterized in that: The mass area ratio of total protein to nitrocellulose in the nanovesicles is 3.75-5 mg:2000-2500 mm 2 .

4. A method for preparing a biosensor for detecting 2-methylisoborneol, comprising: A nitrocellulose dispersion was mixed with an oxidized cellulose dispersion and a 2-hydroxyethyl ferrocenecarboxylate solution, and the mixture was dropped onto the working electrode surface of a screen-printed gold electrode. Then, a nanovesicle solution was dropped and allowed to stand and dry to obtain a biosensor for detecting 2-methylisoborneol.

5. The method for preparing a biosensor for detecting 2-methylisoborneol according to claim 4, 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.

6. The method for preparing a biosensor for detecting 2-methylisoborneol according to claim 4, characterized in that: The oxidized cellulose dispersion is obtained by dispersing oxidized cellulose in deionized water, and the mass volume ratio of oxidized cellulose to deionized water is 0.5-2g:10-100mL.

7. The method for preparing a biosensor for detecting 2-methylisoborneol according to claim 4, characterized in that: The 2-hydroxyethyl ferrocenylcarboxylate solution is obtained by dissolving 2-hydroxyethyl ferrocenylcarboxylate in methanol, and the mass volume ratio of 2-hydroxyethyl ferrocenylcarboxylate to methanol is 1-5g:10-100mL.

8. The method for preparing a biosensor for detecting 2-methylisoborneol according to claim 4, characterized in that: The volume ratio of the nitrocellulose dispersion, the oxidized cellulose dispersion and the 2-hydroxyethyl ferrocenecarboxylate solution is 1-10:1-10:1-10.

9. The method for preparing a biosensor for detecting 2-methylisoborneol according to claim 4, characterized in that: 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 .

10. Use of the biosensor according to claim 1 in detecting 2-methylisoborneol in water samples.

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