A biosensor for detecting urea in tear fluid and preparation and use thereof

Abstract

The application relates to a biosensor for detecting urea in tear fluid and preparation and application thereof, the biosensor takes urea in tear fluid as a biomarker for diagnosing dry eye, an AuAg alloy nanobowl is loaded on a glassy carbon electrode, and an electrochemical biosensor is constructed by combining urease, a standard linear curve of urea concentration and open circuit voltage is established under a three-electrode system, voltage changes before and after adding urea in tear fluid are detected, and the urea concentration is calculated. The tear fluid urea level of a dry eye patient is significantly lower than that of a healthy person, the method can detect the urea content in tear fluid by constructing the sensor, can reflect an ocular surface microenvironment homeostasis in real time, and can provide a reliable basis for accurate diagnosis of dry eye.

Description

Technical Field

[0001] This invention relates to the field of electrochemical biosensors, and in particular to a biosensor for detecting urea in tears, its preparation, and its application. Background Technology

[0002] Dry eye syndrome is a chronic ocular surface disease caused by multiple factors, with impaired tear film homeostasis as its core pathological feature. Symptoms often manifest as eye discomfort and pain, and in severe cases, it can lead to visual impairment and corneal ulcers, seriously threatening patients' visual health and quality of life. As one of the most common ocular surface diseases in clinical practice, the prevalence of dry eye syndrome shows a significant age-related correlation, gradually increasing with age. With the frequent use of mobile phones and air conditioning in modern life, the affected population of dry eye syndrome has further expanded, making accurate diagnosis of dry eye syndrome a key research focus.

[0003] Arginases 1 and 2, and guanidineases exist on the ocular surface, which autonomously synthesize urea by degrading arginine. Urea, as an important component of the tear film, plays a crucial role in maintaining tear film structural stability. Studies have found that the tear urea level in patients with dry eye syndrome is 26.78 ± 5.70 mg·dL. -1 The levels were significantly lower than those in healthy individuals (41.72 ± 6.86 mg·dL). -1 This indicates a close correlation between tear urea levels and dry eye syndrome. Compared to other biological samples, tears offer unique advantages such as simple and non-invasive sample collection, and the ability to reflect dynamic changes in the ocular surface microenvironment in real time. They eliminate the need for complex sampling procedures, effectively reducing patient discomfort during examination. Therefore, using urea as a biomarker for diagnosing dry eye syndrome has promising clinical application prospects and is of great significance for promoting precision diagnosis and treatment of dry eye syndrome.

[0004] Currently, urea detection methods mainly include colorimetric methods, chromatographic methods, non-enzymatic detection, and enzymatic detection. While these methods are applied in different fields, they each have their limitations. Colorimetric methods are easily affected by other substances and have poor stability; chromatographic methods have high sensitivity, but the equipment is expensive, sample pretreatment is complex, and rapid on-site detection is difficult; non-enzymatic detection of urea has a fast response and high stability, but poor selectivity and requires sample pretreatment. Urease detection of urea has high selectivity and catalytic efficiency, but its long-term stability is insufficient. Therefore, developing a highly sensitive, stable, and easy-to-operate urea detection method has become a research hotspot in the field of biological detection.

[0005] AuAg alloy nanobowls, as a key modifying material on the surface of glassy carbon electrodes, play a crucial role in constructing high-performance urea electrochemical sensors. On one hand, the AuAg alloy nanobowls possess a two-dimensional ordered hollow bowl-shaped structure with a large specific surface area, providing ample loading sites for urease and enabling efficient immobilization of enzyme molecules. On the other hand, AuAg alloys exhibit excellent conductivity and chemical stability, significantly accelerating interfacial electron transfer; simultaneously, their good biocompatibility effectively maintains the spatial structural stability of urease, thereby enhancing the sensor's catalytic activity and detection performance for urea. Summary of the Invention

[0006] In view of this, and in light of the shortcomings of the existing technology, the purpose of this application is to provide a biosensor for detecting urea in tears, as well as its preparation and application.

[0007] To address at least one of the aforementioned technical problems, in a first aspect, this application provides an electrode for detecting urea concentration. The electrode includes an electrode substrate and, from the inside out, a nanomaterial film, an enzyme layer, and a capping layer sequentially disposed on the electrode substrate. The nanomaterial is an AuAg alloy nanobowl film.

[0008] On the other hand, this application discloses the preparation and method of an electrode for detecting tear urea concentration, the method comprising:

[0009] After loading nanomaterials onto a glassy carbon electrode, a urease solution is drop-coated and dried, followed by a perfluorosulfonic acid resin solution drop-coated and dried to obtain an electrode for detecting tear urea concentration; the nanomaterial is an AuAg alloy nanobowl film.

[0010] On the other hand, this application discloses an electrochemical biosensor for detecting urea, the sensor comprising a working electrode, a reference electrode, an electrode, and an electrolyte; the working electrode is an electrode prepared by the method described above.

[0011] On the other hand, this application discloses a method for detecting urea concentration, the method comprising: using the above-described electrochemical biosensor to detect urea in a sample to be tested.

[0012] Beneficial effects:

[0013] This invention relates to a biosensor for detecting urea in tears, its preparation, and its application. The biosensor uses urea in tears as a biomarker for diagnosing dry eye syndrome. An electrochemical biosensor is constructed by loading AuAg alloy nanobowls onto a glassy carbon electrode and combining them with urease. A standard linear curve of urea concentration versus open-circuit voltage is established in a three-electrode system. The voltage change before and after adding urea to the tears is detected, and the urea concentration is calculated. Dry eye patients have significantly lower tear urea levels than healthy individuals. This method, by constructing a sensor to detect urea content in tears, can reflect the real-time homeostasis of the ocular surface microenvironment, providing a reliable basis for the accurate diagnosis of dry eye syndrome. Attached Figure Description

[0014] Figure 1 The image shows a SEM image of the AuAg alloy nanobowl film obtained in Example 1, which is a multi-level hollow structure with small nanobowls nested within large nanobowls.

[0015] Figure 2 The standard linear curve of open-circuit voltage change versus urea concentration obtained in Example 2 is shown, with time on the horizontal axis and open-circuit voltage value on the vertical axis. It exhibits a good linear response in the urea concentration range of 0.1-1 mM.

[0016] Figure 3 This is a diagram illustrating the urea detection mechanism of the sensor obtained in Example 3. Urease catalyzes the hydrolysis of urea to NH4. + HCO3 - and OH - This causes a change in the open circuit potential.

[0017] Figure 4 The electrolyte obtained in Example 4 contained 800 μmol·dL -1 The change in open-circuit potential of PBS buffer solution with urea and pH 7.4 is shown on the x-axis as time and the y-axis as open-circuit voltage, which decreased by 16 mV compared to the solution without urea.

[0018] Figure 5 The electrolyte obtained in Example 5 contained 800 μmol·dL -1 The change in open-circuit potential of PBS buffer solution with urea and pH 6 is shown on the x-axis as time and the y-axis as open-circuit voltage, which decreased by 0.9 mV compared to the solution without urea. Detailed Implementation

[0019] The specific embodiments of this application will be further described in detail below with reference to the accompanying drawings. These embodiments are only for illustrating this application and are not intended to limit the scope of the invention. Next, the invention will be explained in more detail through the accompanying drawings and specific examples. These examples are intended to help those skilled in the art to better understand the application of the invention, while it should be understood that these examples do not constitute any limitation on the invention. Furthermore, those skilled in the art will find it entirely possible to make various adjustments and optimizations without departing from the core ideas of the invention. All such adjustments and optimizations should also be considered as part of the protection of this invention.

[0020] To address at least one of the aforementioned technical problems, in a first aspect, this application provides an electrode for detecting urea concentration. The electrode includes an electrode substrate and, from the inside out, a nanomaterial film, an enzyme layer, and a capping layer sequentially disposed on the electrode substrate. The nanomaterial is an AuAg alloy nanobowl film.

[0021] In a specific embodiment, the electrode substrate comprises a glassy carbon electrode.

[0022] In a specific embodiment, the enzyme layer contains urease.

[0023] In a specific embodiment, the overlay layer is used for protection and / or interference resistance; preferably, the overlay layer comprises Nafion.

[0024] On the other hand, this application provides a method for preparing an electrode for detecting tear urea concentration, the method comprising:

[0025] After loading nanomaterials onto a glassy carbon electrode, a urease solution is drop-coated and dried, followed by a perfluorosulfonic acid resin solution drop-coated and dried to obtain an electrode for detecting tear urea concentration; the nanomaterial is an AuAg alloy nanobowl film.

[0026] In a specific embodiment, the concentration of the urease is from 200 U / mg to 300 U / mg. Examples of concentrations of the urease include 50 U / mg, 100 U / mg, 150 U / mg, and 200 U / mg.

[0027] In a specific embodiment, the urease solution contains urease, and the concentration of the urease is 1 mg / mL to 10 mg / mL. Examples of urease concentrations include 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, and 10 mg / mL.

[0028] In a specific embodiment, the amount of urease solution dropped is from 5 μL to 15 μL. For example, the amount of urease solution dropped is 5 μL, 10 μL, or 15 μL.

[0029] In a specific embodiment, the perfluorosulfonic acid resin solution contains perfluorosulfonic acid resin, and the concentration of the perfluorosulfonic acid resin is 0.5 g / 100 mL to 5 g / 100 mL. For example, the concentrations of the perfluorosulfonic acid resin are 0.5 g / 100 mL, 1 g / 100 mL, 2 g / 100 mL, 3 g / 100 mL, 4 g / 100 mL, and 5 g / 100 mL.

[0030] In a specific embodiment, the solvent for the urease solution is a buffer solution. In a specific embodiment, the pH value of the buffer solution is 6.0 to 8.0. For example, the pH values ​​of the buffer solution are 6.0, 7.4, and 8.0.

[0031] The buffer solution has a pH of 6.0 to 8.0, and the buffer solution is selected from one or more of phosphate buffer, tris(hydroxymethyl)aminomethane hydrochloride buffer, and 4-hydroxyethylpiperazine ethanesulfonic acid buffer.

[0032] The pH of the buffer solution is 6.0 to 8.0, and the concentration of the buffer solution is 10 mM to 100 mM. For example, the concentrations of the buffer solutions are 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, and 100 mM.

[0033] On the other hand, this application provides an electrochemical biosensor for detecting urea, the sensor comprising a working electrode, a reference electrode, an electrode, and an electrolyte; the working electrode is the electrode described above or an electrode prepared by any of the methods described above.

[0034] In a specific embodiment, the reference electrode is a silver / silver chloride electrode.

[0035] In a specific embodiment, the electrode is a platinum electrode.

[0036] In a specific embodiment, the electrolyte is a buffer solution. In a specific embodiment, the pH value of the buffer solution is 6.0 to 8.0. Example buffer solutions have pH values ​​of 6.0, 7.4, and 8.0.

[0037] In a specific embodiment, the buffer solution is selected from one or more of phosphate buffer solution, tris(hydroxymethyl)aminomethane hydrochloride buffer solution, and 4-hydroxyethylpiperazine ethanesulfonic acid buffer solution.

[0038] Use of electrodes prepared by any of the methods described above or the electrochemical biosensors described above in the preparation of products for diagnosing dry eye syndrome.

[0039] The buffer solution has a pH of 6.0 to 8.0, and the product further includes urea used to prepare urea standards. The buffer solution has a pH of 6.0 to 8.0, and the urea concentration of the standard curve is 100 μmol / L to 1 mmol / L. Alternatively, the buffer solution may have a pH of 6.0 to 8.0, and the urea concentration may be 500 μmol / L to 800 μmol / L. For example, the urea concentration may be 500 μmol / L or 800 μmol / L.

[0040] In a specific embodiment, the product is used for the detection of a sample to be tested. In a specific embodiment, the sample to be tested is an artificial tear sample or a diluted artificial tear sample.

[0041] The preparation method of AuAg alloy nanobowl thin film includes the following steps: A reaction is carried out at room temperature.

[0042] (1) Use a desiccator as the reaction vessel, place a petri dish containing silver nitrate solution on top of it, and add polystyrene dropwise at the interface.

[0043] After 10-15 minutes, a monolayer polystyrene sphere self-assembled film was completed in an ethanol-water mixed solution of polystyrene spheres. Finely ground dimethylaminoborane powder and nitric acid aqueous solution were placed in the lower layer, and the reaction was carried out for 4-35 hours to obtain a dense silver-polystyrene sphere two-dimensional ordered nanomesh film.

[0044] (2) Place the mixed reaction solution of chloroauric acid solution and disodium hydrogen phosphate solution in a clean petri dish, take a smooth and clean silicon wafer and transfer the silver-polystyrene sphere two-dimensional ordered film prepared in (1) onto the surface of the above reaction solution, react for 0.5-2h, and obtain gold-silver alloy-polystyrene sphere two-dimensional nano-mesh film.

[0045] (3) The silicon wafer loaded with gold-silver alloy-polystyrene film was immersed in tetrahydrofuran solution and reacted for 1.5-3h. The two-dimensional template of polystyrene spheres was removed to obtain AuAg alloy nanobowl film.

[0046] In a specific embodiment, the concentration of the silver nitrate solution mentioned in step (1) is 20-50 mmol / L.

[0047] In a specific embodiment, the volume ratio of ethanol to water in the ethanol-water mixture of polystyrene balls in step (1) is 1:1; the concentration of polystyrene balls is 50-80 g / L.

[0048] In a specific embodiment, the diameter of the polystyrene spheres mentioned in step (1) is 300-400 nm.

[0049] In a specific embodiment, the concentration of the nitric acid aqueous solution in step (1) is 5-10 mol / L.

[0050] In a specific embodiment, the molar ratio of dimethylaminoborane to nitric acid in step (1) is 0.005-0.02.

[0051] In a specific embodiment, the concentration of the chloroauric acid solution in step (2) is 5-15 mmol / L.

[0052] In a specific embodiment, the concentration of the disodium hydrogen phosphate solution in step (2) is 20-200 mmol / L.

[0053] In a specific embodiment, the volume ratio of chloroauric acid solution to disodium hydrogen phosphate solution in step (2) is 1:1-2.

[0054] In a specific embodiment, the AuAg alloy nanobowl film has long-range order, and its smallest structural unit is a hollow nanoparticle of gold-silver alloy with a particle size of 20-50 nanometers.

[0055] In a specific embodiment, the drop volume of the urease solution per unit area is 71 μL / cm². 2 -214μL / cm 2 For example, the drop volume of the urease solution per unit area is 71 μL / cm². 2 71.43 μL / cm 2 90 μL / cm 2 110 μL / cm 2 130 μL / cm 2 142.86 μL / cm 2 143μL / cm 2 160μL / cm 2 180μL / cm 2 500μL / cm 2 214μL / cm 2 214.29 μL / cm 2 .

[0056] In a specific embodiment, the drop volume of the Nafion solution per unit area is 14 μL / cm². 2 -43μL / cm 2 For example, the drop volume of the Nafion is 14 μL / cm². 2 14.28 μL / cm 2 20 μL / cm 2 25 μL / cm 2 28.57 μL / cm 2 29 μL / cm 235 μL / cm 2 40 μL / cm 2 42.86 μL / cm 2 43 μL / cm 2 .

[0057] In a specific embodiment, the amount of urease solution dropped is 5 μL to 15 μL.

[0058] In a specific embodiment, the volume of the Nafion solution dropped is 1 μL to 3 μL.

[0059] In a specific embodiment, the solvent for the perfluorosulfonic acid resin solution is water.

[0060] In a specific embodiment, the concentrations of phosphate buffer, tris(hydroxymethyl)aminomethane hydrochloride, and 4-hydroxyethylpiperazine ethanesulfonic acid in the buffer solution are from 10 mmol / L to 100 mmol / L.

[0061] In a specific embodiment, the electrode substrate area is 0.07 cm². 2 The electrode substrate area refers to the area that can be drop-coated.

[0062] It should be understood that the drop-coating volume per unit area is based on the volume of the electrode substrate (such as a glassy carbon electrode), e.g., "

[0063] The drop volume of the Nafion solution per unit area is 29 μL / cm². 2 "This refers to applying 29 μL of Nafion solution (perfluorosulfonic acid resin solution) per cubic centimeter of electrode substrate."

[0064] The preparation method of AuAg alloy nanobowl thin film includes the following steps: A reaction is carried out at room temperature.

[0065] (1) Take a desiccator as a reaction vessel, place a petri dish containing silver nitrate solution on the upper layer, add ethanol-water mixture of polystyrene spheres at the interface, and the monolayer polystyrene sphere self-assembled membrane is completed after 10-15 min; place finely ground dimethylaminoborane powder and nitric acid aqueous solution on the lower layer, and react for 4-35 h to obtain a dense silver-polystyrene sphere two-dimensional ordered nanomesh film.

[0066] (2) Place the mixed reaction solution of chloroauric acid solution and disodium hydrogen phosphate solution in a clean petri dish. Take a smooth and clean silicon wafer and transfer the silver-polystyrene sphere two-dimensional ordered film prepared in (1) onto the surface of the above reaction solution.

[0067] After 0.5-2 hours, a two-dimensional nanomesh film of gold-silver alloy-polystyrene spheres was obtained.

[0068] (3) The silicon wafer loaded with gold-silver alloy-polystyrene film was immersed in tetrahydrofuran solution and reacted for 1.5-3h. The two-dimensional template of polystyrene spheres was removed to obtain the two-dimensional ordered nanofilm of gold-silver alloy.

[0069] In a specific embodiment, the concentration of the silver nitrate solution mentioned in step (1) is 20-50 mmol / L.

[0070] In a specific embodiment, the volume ratio of ethanol to water in the ethanol-water mixed solution of polystyrene balls described in step (1) is 1:1; the concentration of polystyrene balls is 50-80 g / L.

[0071] In a specific embodiment, the diameter of the polystyrene spheres mentioned in step (1) is 300-400 nm.

[0072] In a specific embodiment, the concentration of the nitric acid aqueous solution in step (1) is 5-10 mol / L.

[0073] In a specific embodiment, the molar ratio of dimethylaminoborane to nitric acid in step (1) is 0.005-0.02.

[0074] In a specific embodiment, the concentration of the chloroauric acid solution in step (2) is 5-15 mmol / L.

[0075] In a specific embodiment, the concentration of the disodium hydrogen phosphate solution in step (2) is 20-200 mmol / L.

[0076] In a specific embodiment, the volume ratio of chloroauric acid solution to disodium hydrogen phosphate solution in step (2) is 1:1-2.

[0077] In a specific embodiment, the two-dimensional gold-silver alloy thin film exhibits long-range order, and its smallest structural unit is a hollow nanoparticle of gold-silver alloy with a particle size of 20-50 nanometers.

[0078] The “AuAg alloy nanobowl film” mentioned in this application refers to the in-situ interface conversion preparation of gold and silver alloy two-dimensional ordered nanofilms prepared by patent CN105543817A (also referred to as AuAg alloy nanobowl film in this application).

[0079] In a specific embodiment, the AuAg alloy nanobowl film was prepared by referring to Example 2 of patent CN105543817A to obtain the AuAg alloy nanobowl film.

[0080] Example: A method for preparing an electrode for detecting tear urea concentration, the method comprising the following steps:

[0081] (1) A desiccator was used as the reaction vessel. A petri dish containing a 20 mmol / L silver nitrate solution was placed flat on the upper layer of the desiccator. 30 μL of a polystyrene sphere ethanol-water (V ethanol:V water = 1:1) mixture was added dropwise to the interface. After a short while, the monolayer polystyrene sphere self-assembled membrane was completed. About 3.5 g of finely ground dimethylaminoborane powder and 5 mL of 5 mol / L nitric acid aqueous solution were placed in the lower layer. After about 25 hours of reaction, a dense silver-polystyrene sphere two-dimensional ordered nanomesh film was obtained.

[0082] (2) Transfer 10 mL of a mixture of chloroauric acid (15 mmol / L) and 15 mL of disodium hydrogen phosphate (200 mmol / L) into a clean petri dish. Take a smooth and clean silicon wafer and transfer the silver-polystyrene sphere two-dimensional ordered film prepared in (1) onto the surface of the above reaction solution. React for about 1 hour to obtain a gold-silver alloy-polystyrene sphere two-dimensional nanomesh film.

[0083] (3) The substrate loaded with gold-silver alloy-polystyrene film was immersed in tetrahydrofuran solution and reacted for 2 hours. The two-dimensional template of polystyrene spheres was removed, which is the AuAg alloy nanobowl film.

[0084] In a specific embodiment, the artificial tears or tear sample contains hydroxypropyl chitosan, protein, sodium chloride, lysozyme, potassium chloride, and calcium chloride. The concentration of the hydroxypropyl chitosan is 0.2 g / 100 ml, the concentration of the protein is 0.05 g / 100 ml, the concentration of the sodium chloride is 1 g / 100 ml, the concentration of the lysozyme is 1 g / 100 ml, the concentration of the potassium chloride is 0.02 g / 100 ml, and the concentration of the calcium chloride is 0.05 g / 100 ml.

[0085] In a specific embodiment, the solvent for the artificial tears is water. For example, the water is deionized water.

[0086] In a specific embodiment, the protein is sodium casein.

[0087] In a specific embodiment, the concentration of the lysozyme is 20000U / 100ml.

[0088] In a specific embodiment, the artificial tears were purchased from Dongguan Chuangwei Testing Instruments Co., Ltd., product number CF-041. Its composition is 0.2% hydroxypropyl chitosan, 0.05% protein, 1% sodium chloride, 1% lysozyme, 0.02% potassium chloride, 0.05% calcium chloride, and the balance being deionized water.

[0089] In a specific embodiment, the dilution factor is 10-70 times. For example, the dilution factors are 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, and 70 times.

[0090] In a specific embodiment, the urea content in the tears of patients with dry eye syndrome is lower than the urea content in the tears of healthy individuals.

[0091] In a specific embodiment, the dry eye syndrome is selected from aqueous dry eye, lipid abnormality dry eye, mucin abnormality dry eye, tear dynamics abnormality dry eye, and mixed dry eye.

[0092] On the other hand, this application provides a method for detecting urea concentration, the method comprising: using the above-described electrochemical biosensor to detect urea in a sample to be tested.

[0093] In a specific embodiment, the method further includes adding different concentrations of urea to the electrolyte to create a standard curve of voltage change versus urea concentration, and calculating the urea concentration using the standard curve.

[0094] In a specific embodiment, the method for preparing the standard curve includes:

[0095] The open-circuit voltage was measured under a three-electrode system and magnetic stirring to obtain a standard linear curve of voltage change versus urea concentration.

[0096] In a specific embodiment, the standard curve contains 5-20 urea concentration points.

[0097] In a specific embodiment, the urea concentration of the standard curve is 100 μmol / L to 1 mmol / L. In another specific embodiment, the urea concentration is 500 μmol / L to 800 μmol / L.

[0098] In a specific embodiment, the sample to be tested contains urea. In a specific embodiment, the urea concentration is 100 μmol / L-1 mmol / L; in a specific embodiment, the urea concentration is 500 μmol / L-800 μmol / L. In a specific embodiment, the pH of the sample to be tested is 6.0-8.0.

[0099] In a specific embodiment, the voltage is the voltage during 100-500 seconds. In a specific embodiment, the voltage is the voltage during 300 seconds.

[0100] In a specific embodiment, the sample to be tested is a tear sample or a diluted tear sample. In a specific embodiment, the dilution factor is 10-70 times. In a specific embodiment, the dilution factor is 40 times.

[0101] On the other hand, this application provides a method for detecting urea for non-therapeutic purposes, the method comprising:

[0102] The sample to be tested is diluted to obtain a diluted solution. The diluted solution is used as an electrolyte, and the above-mentioned biosensor is used to react and detect the voltage change. The concentration of urea in the diluted solution is detected by the voltage change.

[0103] In a specific embodiment, the detection voltage change time is 10s, 30s, 50s, 70s, 90s, 110s, 130s, 150s, 170s, 190s, 210s, 230s, 250s, 270s, or 300s.

[0104] In a specific embodiment, the enzyme or enzyme layer is selected from urease.

[0105] In a specific embodiment, the dilution factor is 10-70 times. In a specific embodiment, the dilution factor is 40 times.

[0106] In a specific embodiment, the detection method further includes constructing a urea standard curve. For example, the concentration of urea in the standard curve shown is 500 μmol·dL. -1 600 μmol·dL -1 700 μmol·dL -1 800 μmol·dL -1 .

[0107] For example, the preparation and detection method of this sensor includes the following steps:

[0108] (1) Prepare AuAg alloy nanobowl film, the method is based on the technical solution disclosed in patent CN105543817A.

[0109] (2) After loading nanomaterials onto a glassy carbon electrode, a solution of urease and perfluorosulfonic acid resin (Nafion) is drop-coated to serve as the working electrode.

[0110] (3) Add urea of ​​different concentrations to the buffer solution, measure the open circuit voltage under a three-electrode system and magnetic stirring, and obtain a standard linear curve of voltage change versus urea concentration;

[0111] (4) Dilute artificial tears with a buffer solution as an electrolyte, measure the voltage change before and after adding urea, and calculate the urea concentration.

[0112] This invention aims to use AuAg alloy nanobowls as electrode modification materials, construct an electrochemical sensor by combining urease, establish a standard linear curve of urea concentration versus voltage change in a three-electrode system, and calculate the urea concentration in the tear fluid based on the voltage change before and after the addition of urea.

[0113] The technical solution adopted in this invention is as follows:

[0114] An electrochemical sensor was prepared for the detection of urea, which can effectively improve the sensitivity of urea detection.

[0115] I. Detection of urea in tears

[0116] (1) A thin film of AuAg alloy nanobowl was loaded on a glassy carbon electrode, and 5 μL to 15 μL of urease solution was dropped onto it. After drying, a 0.5% to 5% Nafion solution was dropped onto it to prepare the sensor.

[0117] (2) Prepare a buffer solution with a concentration of 10 mmol / L to 100 mmol / L in advance, and adjust the pH to 6 to 8;

[0118] (3) A three-electrode system was used, with a silver / silver chloride electrode as the reference electrode, a platinum electrode as the counter electrode, and the above sensor as the working electrode for electrochemical testing. Urea was added to the buffer solution 5 to 20 times under magnetic stirring at 300 rpm to 600 rpm, with the urea concentration ranging from 100 μmol·dL. -1 Up to 1 mmol·dL -1 Record the open-circuit voltage after each addition of urea, and plot a standard linear curve of open-circuit voltage change versus urea concentration;

[0119] (4) Dilute the artificial tears with a buffer solution and record the amount added (100 μmol·dL). -1 Up to 1 mmol·dL -1 Voltage changes from 30s to 300s before and after urea treatment; Preparation of AuAg alloy nanobowls: The method is based on Example 2 of patent CN105543817A, and AuAg alloy nanobowls are obtained.

[0120] The artificial tears were purchased from Dongguan Chuangwei Testing Instruments Co., Ltd., product number CF-041. Its composition is 0.2% hydroxypropyl chitosan, 0.05% protein, 1% sodium chloride, 1% lysozyme, 0.02% potassium chloride, 0.05% calcium chloride, and deionized water as the balance. The protein is sodium casein. The concentration of the lysozyme is 20000 U / 100 ml.

[0121] Nafion, also known as perfluorosulfonic acid resin, has the CAS number 31175-20-9.

[0122] Preparation of 0.5% Nafion solution: Add 200 μL of 5% Nafion to 1.8 mL of deionized water to make the final concentration of Nafion 0.5 g / 100 mL, thus obtaining a 0.5% Nafion solution (also known as a 0.5% Nafion solution).

[0123] Preparation of 5 mg / mL urease solution: Add 5 mg of urease to 1 mL of PBS buffer solution with pH 7.4 to make the enzyme activity of urease 1500 U / mL, and obtain 5 mg / mL urease solution.

[0124] PBS buffer solution with pH 7.4: Add PBS to deionized water to make the concentration of PBS 10mM and the pH of the PBS buffer solution 7.4 to obtain a PBS buffer solution with pH 7.4.

[0125] PBS buffer solution with pH 6.0: Add PBS to deionized water to make the concentration of PBS 10mM and the pH of the PBS buffer solution 6.0 to obtain a PBS buffer solution with pH 6.0.

[0126] Contains 500 μmol·dL -1 Urea-based artificial tears: Dilute the artificial tears 1:40 (v / v) with PBS buffer (pH 7.4), then add urea to achieve a final concentration of 500 μmol·dL. -1 Obtain a solution containing 500 μmol·dL -1 Urea-based artificial tears.

[0127] Contains 800 μmol·dL -1 Urea-based artificial tears: Dilute the artificial tears 1:40 (v / v) with PBS buffer (pH 7.4), then add urea to achieve a final concentration of 800 μmol·dL. -1 Obtained a solution containing 800 μmol·dL -1 Urea-based artificial tears.

[0128] Contains 800 μmol·dL -1 Urea in PBS buffer solution at pH 7.4: Add urea to PBS buffer solution to achieve a final urea concentration of 800 μmol·dL. -1 800 μmol·dL -1 Urea and PBS buffer solution with pH 7.4.

[0129] Contains 800 μmol·dL -1 Urea in PBS buffer solution at pH 6.0: Add urea to the PBS buffer solution to achieve a final urea concentration of 800 μmol·dL. -1 Obtained a solution containing 800 μmol·dL -1 Urea and PBS buffer solution with pH 6.0.

[0130] Example 1

[0131] The prepared AuAg alloy nanobowl film is as follows Figure 1As shown, the nanobowls exhibit a honeycomb-like orderly arrangement, with repeating units being large nanobowls approximately 300 nm in size. The bottom and walls of each large nanobowl are composed of loosely arranged smaller nanobowls, forming a multi-level hollow structure where smaller nanobowls are nested within larger ones. The AuAg alloy nanobowl film was transferred to deionized water, and the film was retrieved upwards using tweezers while holding a glassy carbon electrode. It was dried at room temperature and then drop-coated with 10 μL of 5 mg·mL⁻¹ sodium chloride solution. -1 Urease solution, after drying at 4°C, was drop-coated with 2 μL of 0.5% Nafion solution to modify the electrode. The electrode was then dried at 4°C to obtain the modified electrode (also known as an electrochemical sensor). The urease drop-coating volume was 10 μL, and the electrode area was 0.07 cm². 2 The drop volume per unit area was 142.86 μL / cm². 2 Nafion drop volume: 2 μL; Electrode area: 0.07 cm² 2 The drop volume per unit area was 28.57 μL / cm². 2 (1) Using a silver / silver chloride electrode as the reference electrode, a platinum electrode as the counter electrode, and a modified electrode as the working electrode, the electrolyte was a 10 mM PBS buffer solution with a pH of 7.4. Urea was added to the electrolyte 10 times under magnetic stirring at 300 rpm, so that the urea concentration decreased from 100 μmol·dL in an arithmetic gradient. -1 Change to 1 mmol·dL -1 Record the open-circuit voltage after each addition of urea, and plot a standard linear curve of open-circuit voltage change versus urea concentration.

[0132] (2) A silver / silver chloride electrode was used as the reference electrode, a platinum electrode as the counter electrode, and a modified electrode as the working electrode. The electrolyte contained 500 μmol·dL. -1 For artificial tears containing urea (at a concentration within the urea level range of patients with dry eye), voltage changes were recorded over 300 seconds.

[0133] Example 2

[0134] The AuAg alloy nanobowl film was transferred to deionized water, and the film was lifted upwards using tweezers while holding a glassy carbon electrode. It was dried at room temperature and then drop-coated with 10 μL of 5 mg·mL⁻¹ sodium chloride solution. -1 After drying the urease solution at 4°C, 2 μL of 0.5% Nafion solution (0.5% Nafion solution) was drop-coated onto the modified electrode, which was then dried at 4°C to obtain the modified electrode (also known as an electrochemical sensor).

[0135] (1) Using a silver / silver chloride electrode as the reference electrode, a platinum electrode as the counter electrode, and a modified electrode as the working electrode, the electrolyte was a 10 mmol / L PBS buffer solution with a pH of 7.4. Urea was added to the electrolyte 10 times in an arithmetic gradient under magnetic stirring at 300 rpm, so that the urea concentration was increased from 100 μmol·dL. -1 Change to 1 mmol·dL -1 Record the open-circuit voltage after each addition of urea, and plot a standard linear curve of open-circuit voltage change versus urea concentration, such as... Figure 2 As shown. The sensor exhibits good linear response in the urea concentration range of 0.1–1 mmol / L, with a detection limit of 6.7 μmol·dL. -1 .

[0136] (2) A silver / silver chloride electrode was used as the reference electrode, a platinum electrode as the counter electrode, and a modified electrode as the working electrode. The electrolyte contained 800 μmol·dL. -1 The voltage change was recorded over 300 seconds using artificial tears containing urea (at a concentration within the range of urea levels in healthy individuals).

[0137] Example 3

[0138] The AuAg alloy nanobowl film was transferred to deionized water, and the film was lifted upwards using tweezers while holding a glassy carbon electrode. It was dried at room temperature and then drop-coated with 10 μL of 5 mg·mL⁻¹ sodium chloride solution. -1 After drying the urease solution at 4°C, 2 μL of a 0.5% Nafion solution was drop-coated onto the electrode to modify it. The electrode was then dried at 4°C to obtain the modified electrode.

[0139] (1) A silver / silver chloride electrode was used as the reference electrode, a platinum electrode as the counter electrode, and a modified electrode as the working electrode. The electrolyte contained 800 μmol·dL. -1 A PBS buffer solution containing urea at pH 7.4 was used, and the voltage change was recorded after 300 seconds. The detection mechanism diagram is shown below. Figure 3 As shown, urease is immobilized on AuAg alloy nanobowls and catalyzes the hydrolysis of urea to NH4. + HCO3 - and OH - This causes a change in the open circuit potential.

[0140] Example 4

[0141] The AuAg alloy nanobowl film was transferred to deionized water, and the film was lifted upwards by holding the glassy carbon electrode with tweezers. It was dried at room temperature and then drop-coated with 15 μL of 5 mg·mL⁻¹ sodium chloride solution. -1After drying the urease solution at 4°C, 2 μL of a 0.5% Nafion solution was drop-coated onto the electrode to modify it. The electrode was then dried at 4°C to obtain the modified electrode.

[0142] (1) A silver / silver chloride electrode was used as the reference electrode, a platinum electrode as the counter electrode, and a modified electrode as the working electrode. The electrolyte contained 800 μmol·dL. -1 A PBS buffer solution containing urea at pH 7.4 was used, and the voltage change was recorded after 300 seconds. Figure 4 As shown, it contains 800 μmol·dL -1 The open-circuit potential of the urea electrolyte decreased by 16 mV compared to when no urea was added.

[0143] Example 5

[0144] The AuAg alloy nanobowl film was transferred to deionized water, and the film was lifted upwards using tweezers while holding a glassy carbon electrode. It was dried at room temperature and then drop-coated with 10 μL of 5 mg·mL⁻¹ sodium chloride solution. -1 After drying the urease solution at 4°C, 2 μL of a 0.5% Nafion solution was drop-coated onto the electrode to modify it. The electrode was then dried at 4°C to obtain the modified electrode.

[0145] (1) A silver / silver chloride electrode was used as the reference electrode, a platinum electrode as the counter electrode, and a modified electrode as the working electrode. The electrolyte contained 800 μmol·dL. -1 A PBS buffer solution containing urea at pH 6.0 was used, and the voltage change was recorded after 300 seconds. Figure 5 As shown, it contains 800 μmol·dL -1 The open-circuit potential of the urea electrolyte decreased by 0.9 mV compared to when no urea was added.

[0146] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and substitutions can be made without departing from the technical principles of the present invention, and these improvements and substitutions should also be considered within the scope of protection of the present invention.

Claims

1. An electrode for detecting urea concentration, characterized in that, The electrode includes an electrode substrate and a nanomaterial film, an enzyme layer, and a capping layer sequentially disposed on the electrode substrate from the inside out. The nanomaterial is an AuAg alloy nanobowl film.

2. The electrode as described in claim 1, characterized in that, The nanomaterial film is an AuAg alloy nanobowl film. And / or, the electrode substrate comprises a glassy carbon electrode; And / or, the enzyme layer contains urease; And / or, the overlay is used for protection and / or interference resistance; preferably, the overlay contains Nafion.

3. The method for preparing the electrode according to any one of claims 1 or 2, characterized in that, The method includes: After loading nanomaterials onto a glassy carbon electrode, a urease solution is drop-coated and dried, followed by a perfluorosulfonic acid resin solution drop-coated and dried to obtain an electrode for detecting tear urea concentration; the nanomaterial is an AuAg alloy nanobowl film.

4. The preparation method according to claim 3, characterized in that, The concentration of urease in the urease solution is 200 U / mg to 300 U / mg; And / or, in the urease solution, the concentration of urease is 1 mg / mL to 10 mg / mL; And / or, the drop volume of the urease solution per unit area is 71 μL / cm². 2 -214μL / cm 2 ; And / or, the solvent of the urease solution is a buffer solution; preferably, the pH of the buffer solution is 6.0 to 8.0; more preferably, the buffer solution is selected from one or more of phosphate buffer solution, tris(hydroxymethyl)aminomethane hydrochloride buffer solution, and 4-hydroxyethylpiperazine ethanesulfonic acid buffer solution; And / or, the drop volume of the Nafion solution per unit area is 14 μL / cm². 2 -43μL / cm 2 ; And / or, the perfluorosulfonic acid resin solution contains perfluorosulfonic acid resin, and the concentration of the perfluorosulfonic acid resin is 0.5g / 100mL-5g / 100mL.

5. An electrochemical biosensor for detecting tear uric acid concentration, characterized in that, The sensor includes a working electrode, a reference electrode, a counter electrode, and an electrolyte; the working electrode is the electrode described in claim 1 or 2, or the electrode prepared by the method described in claim 3 or 4.

6. The biosensor as described in claim 5, characterized in that, The reference electrode is a silver / silver chloride electrode; And / or, the counter electrode is a platinum electrode; And / or, the electrolyte is a buffer solution; preferably, the pH of the buffer solution is 6.0 to 8.0; preferably, the buffer solution is selected from one or more of phosphate buffer solution, tris(hydroxymethyl)aminomethane hydrochloride buffer solution, and 4-hydroxyethylpiperazine ethanesulfonic acid buffer solution.

7. Use of the electrode of claim 1 or 2, the electrode prepared by the method of claim 3 or 4, or the electrochemical biosensor of claim 5 or 6 in the preparation of products for detecting urea or for diagnosing dry eye syndrome.

8. The use as described in claim 7, characterized in that, The product also includes urea, which is used to prepare urea standards; preferably, the urea concentration of the standard curve is 100 μmol / L-1 mmol / L; preferably, the urea concentration is 500 μmol / L-800 μmol / L.

9. The use as described in claim 7 or 8, characterized in that, The product is used for the detection of a sample to be tested; preferably, the sample to be tested is a tear sample, a diluted tear sample, an artificial tear sample, or a diluted artificial tear sample; preferably, the dilution factor is 10-70 times; preferably, the dilution factor is 40 times.

10. The use as described in claim 7 or 8, characterized in that, The urea content in the tears of patients with dry eye syndrome is lower than that in the tears of healthy individuals. And / or, the dry eye syndrome is selected from aqueous dry eye, lipid abnormality dry eye, mucin abnormality dry eye, tear dynamics abnormality dry eye, and mixed dry eye.

Images