Preparation method of biosensor for 2-methylisoborneol detection

Abstract

The present invention discloses a preparation method of a biosensor for 2-methylisoborneol detection, belonging to the technical field of biosensors, and specifically relates to a preparation method of a heterologous expression system of olfactory receptors, a preparation method of olfactory receptor cell nanovesicles, a preparation method of a 2-hydroxyethyl ferrocene formate solution, a preparation method of an oxidized cellulose dispersion, a preparation method of a polyvinyl alcohol solution, and a preparation method of a biosensor. The biosensor prepared by the above method has a high electrical signal intensity and high sensitivity.

Description

Technical Field

[0001] The present 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 microorganisms such as cyanobacteria. It is a key earthy-musty odor substance in drinking water, has a strong earthy smell, and can be perceived by the 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] Usually, the content of 2-methylisoborneol in drinking water is extremely low, at the level of nanograms per liter. Currently, gas chromatography-mass spectrometry (GC-MS) or triple quadrupole gas chromatography-tandem mass spectrometry (GC-MS / MS) combined with pretreatment techniques such as liquid-liquid extraction and solid-phase microextraction are usually used for determination. The pretreatment of traditional analytical methods is relatively complex, and the detection instruments are large, making it difficult to meet the needs of on-site detection, especially the ability to respond to sudden odor events is severely insufficient. Electrochemical methods can convert concentration signals into easily measurable electrical signals to achieve the detection of 2-methylisoborneol. And electrochemical sensors are relatively simple to operate, do not require complex sample pretreatment steps, and are easy to deploy and use quickly on-site. However, 2-methylisoborneol itself lacks electrochemical activity and is difficult to directly react on the electrode surface, making it 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, biosensors can specifically recognize target molecules, thereby improving the selectivity of detection. In some previous studies on bionic sensors, 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 lacked specificity and had weak binding ability to odor molecules. Therefore, there is an urgent need for a fast, sensitive, and cost-effective means to detect 2-methylisoborneol in drinking water to improve the efficiency and accuracy of water quality monitoring. Summary of the Invention

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

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

[0007] A biosensor for detecting 2-methylisoborneol, comprising: a screen-printed gold electrode, and a carrier film layer modified on the surface of the working electrode of the screen-printed gold electrode, the carrier film layer containing nanovesicles, the nanovesicles containing olfactory receptor proteins, and the olfactory receptor protein being OR3A4 protein; the carrier film layer includes nitrocellulose, oxidized cellulose, and 2-hydroxyethyl ferrocene formate. Nitrocellulose, oxidized cellulose, and 2-hydroxyethyl ferrocene formate together form a stable film-like structure, enabling the nanovesicles to be evenly distributed therein, and enabling 2-methylisoborneol molecules to diffuse at an appropriate rate to the region of the nanovesicles where the OR3A4 protein is located, while promoting electron transfer, realizing the generation and amplification of signals in the electrochemical biosensor, and improving the accuracy and sensitivity of detection.

[0008] The present invention provides a preparation method of a biosensor for detecting 2-methylisoborneol. Mix a nitrocellulose dispersion liquid with an oxidized cellulose dispersion liquid and a 2-hydroxyethyl ferrocene formate solution, drop the mixture onto the surface of the working electrode of the screen-printed gold electrode, and then drop a nanovesicle solution. After standing and drying, a biosensor for detecting 2-methylisoborneol is obtained.

[0009] Preferably, the nitrocellulose is measured by the area of the nitrocellulose membrane, and the areal mass ratio of nitrocellulose, oxidized cellulose, and 2-hydroxyethyl ferrocene formate in the carrier film layer is 2000 - 2500 mm 2 : 0.05 - 0.2 g: 1 - 5 g.

[0010] Preferably, the mass area ratio of the total protein in the nanovesicles to the nitrocellulose is 3.75 - 5 mg: 2000 - 2500 mm 2 .

[0011] Preferably, the nitrocellulose dispersion liquid 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.

[0012] Preferably, the oxidized cellulose dispersion liquid is obtained by dispersing oxidized cellulose in deionized water. The mass volume ratio of oxidized cellulose to deionized water is 0.5 - 2 g: 10 - 100 mL.

[0013] Preferably, the 2-hydroxyethyl ferrocene formate solution is obtained by dissolving 2-hydroxyethyl ferrocene formate in methanol. The mass volume ratio of 2-hydroxyethyl ferrocene formate to methanol is 1 - 5 g: 10 - 100 mL.

[0014] Preferably, the volume ratio of the nitrocellulose dispersion, the oxidized cellulose dispersion, and the 2-hydroxyethyl ferrocene formate solution is 1-10:1-10:1-10.

[0015] Preferably, the volume ratio of the nitrocellulose dispersion to the nanovesicle solution is 2-10:7.5-37.5.

[0016] 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 .

[0017] More preferably, polyvinyl alcohol can be added during the preparation of the carrier membrane layer in the biosensor for detecting 2-methylisoborneol. The area mass ratio of nitrocellulose, polyvinyl alcohol, oxidized cellulose, and the 2-hydroxyethyl ferrocene formate solution is 2000-2500 mm 2 :0.05-0.2 g:0.05-0.2 g:1-5 g. With the addition of polyvinyl alcohol and the combined action of the carrier membrane layer, the nanovesicles can be evenly distributed on the surface of the working electrode and maintain a relatively fixed position, enabling the biosensor to better adapt to the environment in the living body, reducing the adverse effects on cells, and improving both the biocompatibility and stability of the biosensor.

[0018] The present invention also provides a method for preparing olfactory receptor cell nanovesicles, including:

[0019] Selecting OR3A4 as the human olfactory receptor for 2-methylisoborneol, chemically synthesizing the OR3A4 gene according to the gene sequence information in the Olfactory Receptor DataBase, cloning the OR3A4 gene between the restriction enzyme sites MluI and NotI of the mammalian eukaryotic expression vector pCI, and inserting 2 fusion expression tags, Flag-tag and the first 20 amino acids of human rhodopsin (Rho-tag), between the NheI and EcoRI restriction enzyme sites of the pCI vector to obtain the olfactory receptor expression plasmid;

[0020] Select HEK-293T cells in good growth state, and introduce the plasmid into the cells by liposome transfection method to express olfactory receptor protein. After 12 - 36 h of plasmid transfection, in a serum-free DMEM medium containing cytochalasin B, incubate the HEK293T cells at 36 - 37 °C and 4 - 5% CO₂ for 20 - 40 min. Centrifuge the mixture to remove the remaining cells, take the supernatant and centrifuge to obtain the nanovesicle precipitate. Resuspend it with PBS containing protease inhibitor cocktail to obtain olfactory receptor cell nanovesicles. Quantitatively determine the concentration of olfactory receptor protein in the nanovesicles with a Qubit instrument, and dilute it to the working concentration of 10 - 100 μg / mL with PBS containing protease inhibitor cocktail.

[0021] Preferably, the mass-volume ratio of cytochalasin B to the serum-free DMEM medium is 1 - 10 mg:100 - 500 mL.

[0022] Preferably, the volume ratio of protease inhibitor cocktail to PBS in the PBS containing protease inhibitor cocktail is 1 - 2 mL:100 - 200 mL.

[0023] 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.

[0024] The present invention also provides a preparation method of 2-hydroxyethyl ferrocene formate, including:

[0025] Dissolve ferrocene formic acid in carbon tetrachloride, stir to dissolve under nitrogen protection, continuously pass nitrogen for 20 - 40 min, seal the reaction system and add oxalyl chloride, stir and react at room temperature for 6 - 10 h, rotary evaporate to remove the carbon tetrachloride solvent and unreacted oxalyl chloride to obtain ferrocene carbonyl chloride;

[0026] Mix and dissolve ethylene glycol and dichloromethane, add triethylamine, place it in an ice-water bath under nitrogen protection and stir to react for 15 - 45 min. Dissolve ferrocene carbonyl chloride in dichloromethane and add it to the reaction system, remove the ice-water bath, stir and react at room temperature for 12 - 36 h, wash with saturated brine, and rotary evaporate to remove the remaining solvent to obtain 2-hydroxyethyl ferrocene formate.

[0027] Preferably, the mass-volume ratio of ferrocene formic acid to carbon tetrachloride is 1 - 5 g:20 - 100 mL.

[0028] Preferably, the mass-volume ratio of ferrocene formic acid to oxalyl chloride is 1 - 5 g:1 - 5 mL.

[0029] Preferably, the mass-volume ratio of ethylene glycol to dichloromethane is 1 - 5 g:10 - 50 mL.

[0030] Preferably, the mass-volume ratio of ethylene glycol to triethylamine is 1-5 g: 0.5-2.5 mL.

[0031] Preferably, the mass ratio of ethylene glycol to ferrocenecarbonyl chloride is 1-5: 4-20.

[0032] Preferably, the mass-volume ratio of ferrocenecarbonyl chloride to dichloromethane is 4-20 g: 10-50 mL.

[0033] The present invention also provides a method for preparing an oxidized cellulose slurry, comprising:

[0034] Disperse microcrystalline cellulose in a solution containing 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO) and sodium bromide solution, add sodium hypochlorite, adjust the pH to 9.8-10.2, heat to 35-45 °C and soak for 2-4 h, and wash the TEMPO-oxidized cellulose with deionized water; redisperse the TEMPO-oxidized cellulose in deionized water and treat it with an ultrasonic cell disruptor to obtain an oxidized cellulose slurry.

[0035] Preferably, the mass ratio of microcrystalline cellulose, TEMPO to sodium bromide is 1-10: 0.016-0.16: 0.1-1.

[0036] Preferably, the mass ratio of microcrystalline cellulose to sodium hypochlorite is 1-10: 0.74-7.4.

[0037] Preferably, the mass-volume ratio of TEMPO-oxidized cellulose to deionized water is 1-10 g: 10-100 mL.

[0038] Preferably, the power of the ultrasonic cell disruptor is 300 W and the treatment time is 10-20 min.

[0039] The present invention also provides a method for preparing a biosensor, comprising:

[0040] Disperse a nitrocellulose membrane in methanol to obtain a nitrocellulose dispersion; dissolve 2-hydroxyethyl ferrocenecarboxylate in methanol to obtain a 2-hydroxyethyl ferrocenecarboxylate solution; disperse the oxidized cellulose slurry in deionized water, heat and stir at 75-85 °C until completely dispersed to form a homogeneous solution; mix the nitrocellulose dispersion, the oxidized cellulose dispersion and the 2-hydroxyethyl ferrocenecarboxylate solution, adjust the pH value to 7.0-7.2, and stir at room temperature to obtain a mixed solution; drop the mixed solution onto the working electrode on the surface of a screen-printed gold electrode, and leave it at room temperature until the methanol and water evaporate and dry; drop a nanovesicle solution containing olfactory receptor protein onto the surface of the gold electrode; leave the modified microelectrode at room temperature for 1-3 h to obtain a biosensor.

[0041] 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.

[0042] Preferably, the mass - volume ratio of 2 - hydroxyethyl ferrocene formate to methanol is 1 - 5 g: 10 - 100 mL.

[0043] Preferably, the mass - volume ratio of oxidized cellulose slurry to deionized water is 0.5 - 2 g: 10 - 100 mL.

[0044] Preferably, the volume ratio of the nitrocellulose dispersion, the oxidized cellulose dispersion to the 2 - hydroxyethyl ferrocene formate solution is 1 - 10: 1 - 10: 1 - 10.

[0045] Preferably, the volume ratio of the nitrocellulose dispersion to the nanovesicle solution is 2 - 10: 7.5 - 37.5.

[0046] 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 .

[0047] The present invention discloses a biosensor prepared by the above - mentioned method.

[0048] Since the present invention constructs a heterologous expression system capable of highly expressing an olfactory receptor protein with specific recognition ability for 2 - methylisoborneol, and provides a preparation method for olfactory receptor cell nanovesicles. The biosensor is modified with 2 - hydroxyethyl ferrocene formate, oxidized cellulose and polyvinyl alcohol, thus having the following beneficial effects: The carrier membrane layer provides a stable immobilization environment for the nanovesicles containing olfactory receptors and promotes electron transfer, increasing the biocompatibility and stability of the biosensor. Therefore, the present invention is a biosensor with strong electrical signals and its preparation method. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 is a schematic structural diagram of the biosensor;

[0050] Figure 2 is a test result graph of the vesicle shedding rate of the biosensor;

[0051] Figure 3 is a test result graph of the electrical signal intensity of the biosensor. DETAILED DESCRIPTION OF THE INVENTION

[0052] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with 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.

[0053] First, the concepts involved in this application will be described in conjunction with the accompanying drawings. It should be noted here that the descriptions of the following concepts are only for making the content of this application easier to understand and do not represent a limitation on the protection scope of this application; at the same time, without conflict, the embodiments and features in the embodiments of this application can be combined with each other. This application will be described in detail below with reference to the accompanying drawings and in conjunction with the embodiments.

[0054] Example 1:

[0055] A method for preparing a biosensor for detecting 2-methylisoborneol, comprising:

[0056] Construction of an olfactory receptor heterologous expression system: Select OR3A4 as the human olfactory receptor for 2-methylisoborneol and HEK-293T cells as host cells. According to the gene sequence information in the Olfactory Receptor DataBase, chemically synthesize the OR3A4 gene, clone the OR3A4 gene between the restriction enzyme sites MluI and NotI of the mammalian eukaryotic expression vector pCI, insert two fusion expression tags, Flag-tag and the first 20 amino acids of human rhodopsin (Rho-tag), between the NheI and EcoRI restriction enzyme sites of the pCI vector to obtain an olfactory receptor expression plasmid. Select HEK-293T cells with good growth status and use the liposome transfection method to introduce the plasmid into the cells to express the olfactory receptor protein.

[0057] Preparation of olfactory receptor cell nanovesicles: 24 hours after plasmid transfection, incubate HEK293T cells in serum-free DMEM medium containing 20 μg / mL cytochalasin B at 37 °C and 5% CO2 for 30 min. Centrifuge the mixture at 500×g for 10 min, take the supernatant and centrifuge it at 15000×g for 30 min to obtain a nanovesicle precipitate. Resuspend it with PBS containing a protease inhibitor cocktail to obtain olfactory receptor cell nanovesicles. Quantitatively measure the concentration of olfactory receptor protein in the nanovesicles with a Qubit instrument and dilute it to a working concentration of 100 μg / mL.

[0058] Preparation of 2 - hydroxyethyl ferrocene formate: Under nitrogen protection, 1.15 g of ferrocene formic acid was mixed with 25 mL of carbon tetrachloride and stirred until the ferrocene formic acid was completely dissolved. After continuously passing nitrogen for 30 min, the system was sealed. 1.2 mL of oxalyl chloride was added, and after stirring and reacting at room temperature for 8 h, the carbon tetrachloride solvent and unreacted oxalyl chloride were removed by rotary evaporation to obtain ferrocene carbonyl chloride; 3.1 g of ethylene glycol and 25 mL of dry dichloromethane were mixed and stirred, 1.4 mL of triethylamine was added, and the mixture was placed in an ice - water bath and stirred under nitrogen protection for 30 min; 12 g of the prepared ferrocene carbonyl chloride was dissolved in 20 mL of dichloromethane and added to the reaction system. After removing the ice - water bath and stirring at room temperature for 24 h, it was washed with saturated brine, and the remaining solvent was removed by rotary evaporation to obtain 2 - hydroxyethyl ferrocene formate.

[0059] Preparation of oxidized cellulose slurry: 1 g of microcrystalline cellulose was dispersed in a solution of 0.016 g of 2,2,6,6 - tetramethylpiperidine - 1 - oxyl radical (TEMPO) and 0.1 g of sodium bromide. 0.74 g of sodium hypochlorite was added, the pH was adjusted to 10.0, and after heating to 40 °C and soaking for 3 h, the TEMPO - oxidized cellulose was washed with deionized water; 1 g of TEMPO - oxidized cellulose was redispersed in 10 mL of deionized water and treated with an ultrasonic cell disruptor for 15 min to obtain oxidized cellulose slurry, which was stored frozen and sealed.

[0060] Preparation of biosensor: The nitrocellulose membrane was dispersed in methanol to obtain a nitrocellulose dispersion; 2.3 g of 2 - hydroxyethyl ferrocene formate was dissolved in 10 mL of methanol to obtain a 2 - hydroxyethyl ferrocene formate solution; 1 g of oxidized cellulose was dispersed in 100 mL of deionized water, heated to 80 °C, and continuously stirred to form an oxidized cellulose dispersion; 1 mL of nitrocellulose dispersion, 1 mL of oxidized cellulose dispersion, and 1 mL of 2 - hydroxyethyl ferrocene formate solution were mixed, the pH value was adjusted to 7.0, and the mixture was stirred at room temperature to obtain a mixed solution; 12 μL of the mixed solution was dropped onto the working electrode surface of the screen - printed gold electrode, and it was left standing at room temperature until the methanol and water evaporated and dried; 15 μL of the nanovesicle solution containing olfactory receptor protein was dropped onto the working electrode surface of the screen - printed gold electrode, and it was left standing at room temperature for 2 h to obtain the biosensor. The pore size of the nitrocellulose membrane is 0.45 μm, and the area - volume ratio of the nitrocellulose membrane dispersed in methanol is 1 mm 2 : 5 μL; the area of the working electrode on the screen - printed gold electrode surface is 20 mm 2 , when the mixed solution is dropped onto the working electrode of the screen - printed gold electrode surface, it is required to only cover the working electrode and not touch the counter electrode and the reference electrode; when dropping the nanovesicle solution, it is required to only cover the working electrode and not touch the counter electrode and the reference electrode.

[0061] Example 2:

[0062] Preparation method of a biosensor for detecting 2-methylisoborneol

[0063] This example is different from Example 1 only in the preparation of the biosensor.

[0064] Preparation of the biosensor: Disperse the nitrocellulose membrane in methanol to obtain a nitrocellulose dispersion; dissolve 2.3 g of 2-hydroxyethyl ferrocene formate in 10 mL of methanol to obtain a 2-hydroxyethyl ferrocene formate solution; disperse 2 g of oxidized cellulose in 100 mL of deionized water, heat to 80 °C, and continuously stir to form an oxidized cellulose dispersion; mix 1 mL of the nitrocellulose dispersion, 1 mL of the oxidized cellulose dispersion, and 1 mL of the 2-hydroxyethyl ferrocene formate solution, adjust the pH value to 7.0, and stir at room temperature to obtain a mixed solution; drop 12 μL of the mixed solution onto the surface of the working electrode on the surface of the screen-printed gold electrode, and let it stand at room temperature until the methanol and water evaporate and dry; drop 15 μL of the nanovesicle solution containing olfactory receptor protein onto the surface of the gold electrode, and let it stand at room temperature for 2 h to obtain the biosensor. The pore size of the nitrocellulose membrane is 0.45 μm, and the area-volume ratio of the nitrocellulose membrane dispersed in methanol is 1 mm 2 : 5 μL; the area of the working electrode on the surface of the screen-printed gold electrode is 20 mm 2 When the mixed solution 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 touch the counter electrode and the reference electrode; when dropping the nanovesicle solution, it is required to only cover the working electrode and not touch the counter electrode and the reference electrode.

[0065] Example 3:

[0066] Preparation method of a biosensor for detecting 2-methylisoborneol

[0067] This example is different from Example 1 only in the preparation of the biosensor.

[0068] Preparation of biosensor: Disperse the nitrocellulose membrane in methanol to obtain a nitrocellulose dispersion; dissolve 2.3 g of 2-hydroxyethyl ferrocene formate in 10 mL of methanol to obtain a 2-hydroxyethyl ferrocene formate solution; disperse 1 g of oxidized cellulose in 100 mL of deionized water, heat to 80 °C, and continuously stir to form an oxidized cellulose dispersion; dissolve 1 g of polyvinyl alcohol in 100 mL of deionized water, heat to 80 °C, and stir until completely dissolved to obtain a polyvinyl alcohol solution; mix 1 mL of polyvinyl alcohol solution, 1 mL of nitrocellulose dispersion, 1 mL of oxidized cellulose dispersion, and 1 mL of 2-hydroxyethyl ferrocene formate solution, adjust the pH value to 7.0, and stir at room temperature to obtain a mixed solution; drop 16 μL of the mixed solution onto 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 15 μL of the nanovesicle solution containing olfactory receptor protein onto the gold electrode surface, and let it stand at room temperature for 2 h to obtain the biosensor. The pore size of the nitrocellulose membrane is 0.45 μm, and the area-volume ratio of the nitrocellulose membrane dispersed in methanol is 1 mm 2 :5 μL; the area of the working electrode on the screen-printed gold electrode surface is 20 mm 2 , when the mixed solution is dropped onto the working electrode of the screen-printed gold electrode surface, it is required to only cover the working electrode and not touch the counter electrode and the reference electrode; when dropping the nanovesicle solution, it is required to only cover the working electrode and not touch the counter electrode and the reference electrode.

[0069] Example 4:

[0070] A method for preparing a biosensor for detecting 2-methylisoborneol,

[0071] Compared with Example 1, the difference in this example lies only in the preparation of the biosensor.

[0072] Preparation of biosensor: The nitrocellulose membrane was dispersed in methanol to obtain a nitrocellulose dispersion; 2.3 g of 2-hydroxyethyl ferrocene formate was dissolved in 10 mL of methanol to obtain a 2-hydroxyethyl ferrocene formate solution; 1 g of oxidized cellulose was dispersed in 100 mL of deionized water and heated to 80 °C, and continuously stirred to form an oxidized cellulose dispersion; 2 g of polyvinyl alcohol was dissolved in 100 mL of deionized water and heated to 80 °C and stirred until completely dissolved to obtain a polyvinyl alcohol solution; 1 mL of the polyvinyl alcohol solution, 1 mL of the nitrocellulose dispersion, 1 mL of the oxidized cellulose dispersion and 1 mL of the 2-hydroxyethyl ferrocene formate solution were mixed, and the pH value was adjusted to 7.0, and stirred at room temperature to obtain a mixed solution; 16 μL of the mixed solution was dropped onto the working electrode surface of the screen-printed gold electrode surface, and left standing at room temperature until methanol and water volatilized and dried; 15 μL of the nanovesicle solution containing olfactory receptor protein was dropped onto the gold electrode surface, and left standing at room temperature for 2 h to obtain a biosensor. The pore size of the nitrocellulose membrane was 0.45 μm, and the area-to-volume ratio of the nitrocellulose membrane dispersed in methanol was 1 mm 2 : 5 μL; the working electrode area on the screen-printed gold electrode surface was 20 mm 2 When the mixed solution was dropped onto the working electrode of the screen-printed gold electrode surface, it was required to only cover the working electrode and not touch the counter electrode and the reference electrode; when dropping the nanovesicle solution, it was required to only cover the working electrode and not touch the counter electrode and the reference electrode.

[0073] Comparative Example 1:

[0074] This comparative example was different from Example 1 only in the preparation of the biosensor.

[0075] Preparation of biosensor: The nitrocellulose membrane was dispersed in methanol to obtain a nitrocellulose dispersion; 2.3 g of 2-hydroxyethyl ferrocene formate was dissolved in 10 mL of methanol to obtain a 2-hydroxyethyl ferrocene formate solution; 1 mL of the nitrocellulose dispersion and 1 mL of the 2-hydroxyethyl ferrocene formate solution were mixed, and the pH value was adjusted to 7.0, and stirred at room temperature to obtain a mixed solution; 8 μL of the mixed solution was dropped onto the working electrode surface of the screen-printed gold electrode surface, and left standing at room temperature until methanol and water volatilized and dried; 15 μL of the nanovesicle solution containing olfactory receptor protein was dropped onto the working electrode surface of the screen-printed gold electrode surface, and left standing at room temperature for 2 h to obtain a biosensor. The pore size of the nitrocellulose membrane was 0.45 μm, and the area-to-volume ratio of the nitrocellulose membrane dispersed in methanol was 1 mm 2 : 5 μL; the working electrode area on the screen-printed gold electrode surface was 20 mm 2When the mixed solution is added dropwise to the working electrode on the surface of the screen-printed gold electrode, it is required to only cover the working electrode and not touch the counter electrode and the reference electrode; when adding the nanovesicle solution dropwise, it is required to only cover the working electrode and not touch the counter electrode and the reference electrode.

[0076] Comparative Example 2:

[0077] The difference between this comparative example and Example 1 lies only in the preparation of the biosensor.

[0078] Preparation of the biosensor: The nitrocellulose membrane was dispersed in methanol to obtain a nitrocellulose dispersion; 1 g of oxidized cellulose was dispersed in 100 mL of deionized water and heated to 80 °C, and continuously stirred to form an oxidized cellulose dispersion; 1 mL of the nitrocellulose dispersion was mixed with 1 mL of the oxidized cellulose dispersion, the pH value was adjusted to 7.0, and stirred at room temperature to obtain a mixed solution; 8 μL of the mixed solution was added dropwise to the surface of the working electrode on the surface of the screen-printed gold electrode, and left standing at room temperature until the methanol and water volatilized and dried; 15 μL of the nanovesicle solution containing the olfactory receptor protein was added dropwise to the surface of the working electrode on the surface of the screen-printed gold electrode, and left standing at room temperature for 2 h to obtain the biosensor. The pore size of the nitrocellulose membrane is 0.45 μm, and the area-volume ratio of the nitrocellulose membrane dispersed in methanol is 1 mm 2 : 5 μL; the area of the working electrode on the surface of the screen-printed gold electrode is 20 mm 2 When the mixed solution is added dropwise to the working electrode on the surface of the screen-printed gold electrode, it is required to only cover the working electrode and not touch the counter electrode and the reference electrode; when adding the nanovesicle solution dropwise, it is required to only cover the working electrode and not touch the counter electrode and the reference electrode.

[0079] Comparative Example 3:

[0080] The difference between this comparative example and Example 1 lies only in the preparation of the biosensor.

[0081] Preparation of the biosensor: The nitrocellulose membrane was dispersed in methanol to obtain a nitrocellulose dispersion; 4 μL of the nitrocellulose dispersion was added dropwise to the surface of the working electrode on the surface of the screen-printed gold electrode, and left standing at room temperature until the methanol and water volatilized and dried; 15 μL of the nanovesicle solution containing the olfactory receptor protein was added dropwise to the surface of the working electrode on the surface of the screen-printed gold electrode, and left standing at room temperature for 2 h to obtain the biosensor. The pore size of the nitrocellulose membrane is 0.45 μm, and the area-volume ratio of the nitrocellulose membrane dispersed in methanol is 1 mm 2 : 5 μL; the area of the working electrode on the surface of the screen-printed gold electrode is 20 mm 2When the mixed liquid is added dropwise to 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 adding the nanovesicle solution dropwise, it is required to only cover the working electrode and not contact the counter electrode and the reference electrode.

[0082] The structural schematic diagram of the biosensor prepared in Example 1 of the present invention is as Figure 1 shown, where the working electrode is a screen-printed gold electrode modified with nanovesicles containing olfactory receptor proteins, the carrier membrane layer includes 2-hydroxyethyl ferrocene formate, oxidized cellulose, polyvinyl alcohol and nitrocellulose, the counter electrode is a screen-printed gold electrode, and the reference electrode is silver / silver chloride.

[0083] Test Example 1: Vesicle shedding rate

[0084] Test samples: Biosensors prepared by the methods of each example and comparative example.

[0085] Test method: Use a Qubit instrument to measure the concentration of olfactory receptor protein (C0) in the initially adsorbed nanovesicles on the surface of the biosensor. Place the biosensor in drinking water at room temperature and oscillate for 24 h, then use the Qubit instrument again to measure the concentration of olfactory receptor protein (C1) in the remaining nanovesicles on the surface of the biosensor. According to the formula: vesicle shedding rate = (C0 - C1) / C0 × 100%, calculate the vesicle shedding rate.

[0086] The test results of the vesicle shedding rate prepared by the present invention are as Figure 2As shown, the vesicle shedding rate of Example 1 was 11.7%, that of Example 2 was 13.2%, the vesicle shedding rate of Example 3 was the lowest, at 7.8%, and the vesicle shedding rate of Example 4 was 10.3%. It can be seen from Example 1 to Example 3 that the optimization of the preparation process, especially after adding polyvinyl alcohol, the vesicle shedding rate decreased, indicating that polyvinyl alcohol helps to enhance the binding force between the vesicles and the electrode surface. Compared with Example 1, Example 2, and compared with Example 3, Example 4 changed the dosage and ratio of each component during the preparation of the biosensor, resulting in an increase in the vesicle shedding rate, indicating that appropriate solution dosage has a positive effect on vesicle stability. In Comparative Example 1, without using the oxidized cellulose dispersion, the vesicle shedding rate reached 24.3%, which was significantly higher, indicating that oxidized cellulose plays an important role in the stable fixation of vesicles. In Comparative Example 2, without using 2-hydroxyethyl ferrocene formate solution, the vesicle shedding rate was 23.0%, which was also significantly higher than that of Example 3, indicating that 2-hydroxyethyl ferrocene formate helps to enhance the binding stability between the vesicles and the electrode. In Comparative Example 3, neither 2-hydroxyethyl ferrocene formate nor oxidized cellulose dispersion was used, and its vesicle shedding rate was 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 shows that after modifying the nano-vesicles with 2-hydroxyethyl ferrocene formate, oxidized cellulose and polyvinyl alcohol according to the method of the present invention, the vesicle shedding rate has been significantly reduced. The overall vesicle shedding rate of the biosensor prepared by the present invention is in the range of 7-14%, significantly improving the stability of the biosensor.

[0087] Test Example 2: Electric signal intensity

[0088] Test samples: Biosensors prepared by the methods of each example and comparative example.

[0089] Test method: Prepare an electrolyte solution containing 5 mmol / L potassium ferricyanide, 5 mmol / L potassium ferrocyanide and 0.1 mol / L potassium chloride with ultrapure water. Connect the microelectrode modified with nano-vesicles to an electrochemical workstation, drop 100 μL of the electrolyte solution on the surface of the printed electrode, ensure that the working electrode, reference electrode and counter electrode are completely immersed, and perform cyclic voltammetry scanning. The parameter settings are a scanning speed of 0.05 V / s, a scanning range of 0.7 V to -0.3 V, and 2 scanning cycles to measure the peak current signal intensity of different biosensors.

[0090] The present invention uses the redox system of potassium ferricyanide - potassium ferrocyanide to characterize the electrochemical detection effect of the biosensor. The test results of the peak current signal intensity are as Figure 3As shown, the oxidation peak current potential of Example 1 was 6.7 μA, that of Example 2 was 6.8 μA, the oxidation peak current potential of Example 3 was the highest, reaching 9.0 μA, and the current of Example 4 was 8.5 μA. From Example 1 to Example 3, the electrical signal intensity gradually increased, which was closely related to the changes in the dosage and ratio of the oxidized cellulose dispersion, 2-hydroxyethyl ferrocene formate solution, and polyvinyl alcohol used in the preparation of the biosensor in each example. After optimizing the solution ratio and adding polyvinyl alcohol in Example 3, the current reached the highest value of 9.0 μA, indicating that polyvinyl alcohol synergistically interacted with other components, significantly enhancing the electrical signal response intensity of the biosensor. On the basis of Example 3, Example 4 changed the solution dosage, and the electrical signal intensity slightly decreased to 8.5 μA, once again reflecting the important influence of the dosage and ratio of each component on the performance of the biosensor. In Comparative Example 1, the oxidized cellulose dispersion was not used, and its current was only 4.1 μA, far lower than that of Example 3, indicating that oxidized cellulose had a positive effect on enhancing the electrical signal. In Comparative Example 2, the 2-hydroxyethyl ferrocene formate solution was not used, and the current was 3.9 μA, which was also significantly lower than that of Example 3, indicating that 2-hydroxyethyl ferrocene formate played an important role in enhancing the electrical signal intensity. In Comparative Example 3, neither the 2-hydroxyethyl ferrocene formate nor the oxidized cellulose dispersion was used, and the current was the lowest, only 3.2 μA, further confirming the key role of these two substances in increasing the electrical signal intensity of the biosensor. Compared with Comparative Examples 1-3, Experimental Example 3 showed that after modifying the nanovesicles with 2-hydroxyethyl ferrocene formate, oxidized cellulose, and polyvinyl alcohol according to the method of the present invention, the peak current signal intensity of the biosensor was significantly increased. The biosensor prepared by the present invention had the highest peak current signal intensity, and the current was between 6-9 μA, significantly improving the electrical signal response intensity detected by the biosensor.

[0091] 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 changes or modifications to other equivalent embodiments, but should still be regarded as the same technology or embodiment as the present invention in essence.

[0092] In this text, specific examples are used to elaborate on the principles and implementation modes of the present application. The description of the above embodiments is only for helping to understand the method of the present application and its core idea. The above is only the preferred implementation mode of the present application. It should be noted that due to the limited nature of literal expression and the objectively infinite specific structures, for those of ordinary skill in the art, without departing from the principles of the present application, several improvements, embellishments or changes can be made, or the above technical features can be combined in an appropriate manner; these improvements, embellishments, changes or combinations, or directly applying the inventive concept and technical solution to other occasions without improvement, shall all be regarded as the protection scope of the present 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 includes nitrocellulose, oxidized cellulose and 2-hydroxyethyl ferrocene formate; 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; The mass area ratio of total protein to nitrocellulose in the nanovesicles is 3.75-5 mg:2000-2500 mm 2 .

2. A method for preparing a biosensor for detecting 2-methylisoborneol according to claim 1, 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 added, and the mixture was allowed to stand and dry to obtain a biosensor for detecting 2-methylisoborneol.

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

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

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

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

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

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

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