Rapid urea concentration detection method based on photoelectrochemical analysis and application thereof

Abstract

The invention relates to a rapid urea concentration detection method based on photoelectrochemical analysis and application of the rapid urea concentration detection method based on photoelectrochemical analysis, and the rapid urea concentration detection method based on photoelectrochemical analysis comprises the following steps: (1) preparing a CDs / C3N4 / ITO electrode; (2) adding a urease solution, a urease reaction buffer solution and urea solutions with different concentrations into a 96-well plate, and incubating; (3) preparing a CDs / C3N4 / ITO electrode for adsorbing methylene blue; (4) carrying out photoelectrochemical detection on the urea concentration; (5) drawing a standard curve contrast diagram; and (6) detecting the concentration of the urea solution with unknown concentration. The detection method is used for detecting the urea concentration in human body fluid or environmental water. The urea concentration rapid detection method can be used for preparing a urea concentration detection sensor. The detection method disclosed by the invention has the advantages of excellent selectivity, good repeatability and the like when being used for instantly detecting the urea concentration in human body fluid or environment.

Description

Technical Field

[0001] The invention relates to a method for rapid detection of urea concentration and application thereof, and in particular to a method for rapid detection of urea concentration based on photoelectrochemical analysis and application thereof. Background Art

[0002] Urea is the main component of nitrogen fertilizer. The wastewater discharged during its production and the farmland runoff during its use will cause pollution to rivers, lakes and groundwater sources, becoming an important source of ammonia nitrogen and nitrate pollutants in water bodies. Eutrophication is becoming increasingly serious, and it also causes groundwater pollution. For example, a large amount of nitrogen fertilizer entering the water body provides rich nitrogen nutrition conditions for the rapid proliferation of "red tide organisms", which has become one of the main factors causing red tides. The occurrence of red tides destroys the marine ecosystem, poisons or kills fish and shellfish, and endangers human health through the food chain.

[0003] Urea is an important metabolite in the human liver. The disorder of its level in body fluid components is closely related to a variety of diseases such as heart and kidney failure. Therefore, the accurate detection of urea concentration in human body fluids is crucial in clinical diagnosis. Traditional methods for urea detection mainly include gas chromatography, calorimetry and fluorescence analysis. Although these methods are reliable, they all require high costs, large instruments and complex operations, and are not suitable for on-site monitoring. Summary of the invention

[0004] The technical problem to be solved by the present invention is to overcome the deficiencies of the prior art and provide a method for rapid detection of urea concentration based on photoelectrochemical analysis, which is low in cost, suitable for on-site monitoring, rapid and easy to use, and its application.

[0005] The technical solution adopted by the present invention to solve the technical problem is a method for rapid detection of urea concentration based on photoelectrochemical analysis, comprising the following steps:

[0006] (1) dropping a uniformly dispersed CDs / C3N4 solution onto an indium tin oxide glass sheet and drying it for later use to form a CDs / C3N4 / ITO electrode;

[0007] (2) adding urease solution, urease reaction buffer and urea solutions of different concentrations into a 96-well plate and incubating at a certain temperature for a period of time;

[0008] (3) adding methylene blue solution to the 96-well plate of step (2), placing the CDs / C3N4 / ITO electrode obtained in step (1) into the well plate for adsorption for a period of time, then taking it out, washing the electrode with PBS washing solution, and obtaining a CDs / C3N4 / ITO electrode adsorbing methylene blue;

[0009] (4) In a three-electrode system, the CDs / C3N4 / ITO electrode adsorbing methylene blue was used as the working electrode to perform photoelectrochemical detection of urea concentration;

[0010] (5) drawing a standard curve comparison chart based on the urea solutions of different concentrations corresponding to the different photocurrent values ​​obtained in step (4);

[0011] (6) Repeat steps (1) to (4) for a urea solution of unknown concentration to be tested to obtain a photocurrent value, and compare the photocurrent value with the standard curve reference chart described in step (5) to determine the urea concentration of the solution to be tested.

[0012] Preferably, the steps include:

[0013] (1) Drop 20 μL of evenly dispersed CDs / C3N4 solution on an indium tin oxide glass sheet and dry it for later use to form a CDs / C3N4 / ITO electrode;

[0014] (2) Add 50 μL of urease solution (preferably containing 6 U mL urease) -1 ), 90 μL urease reaction buffer and 100 μL urea solution of different concentrations (0-60 mM) were added to a 96-well plate and incubated at 37°C for 30 min;

[0015] (3) adding 10 μL of methylene blue solution to each of the 96-well plates of step (2), placing the CDs / C3N4 / ITO electrode obtained in step (1) into the well plate for adsorption (preferably for 8-12 min, more preferably for 10 min), then taking it out, and washing the electrode three times with PBS washing solution to obtain a CDs / C3N4 / ITO electrode adsorbing methylene blue;

[0016] (4) In a three-electrode system, the CDs / C3N4 / ITO electrode adsorbing methylene blue was used as the working electrode to perform photoelectrochemical detection of urea concentration;

[0017] (5) drawing a standard curve comparison chart according to the urea solutions of different concentrations corresponding to the different photocurrent values ​​obtained in step (4);

[0018] (6) Repeat steps (1) to (4) for a urea solution of unknown concentration to be tested to obtain a photocurrent value, and compare the photocurrent value with the standard curve reference chart described in step (5) to determine the urea concentration of the solution to be tested.

[0019] The preparation method of CDs / C3N4 composite material is a prior art, and can also be prepared by the following method, including the following steps: (1.1) Synthesis of carbon quantum dots (CDs): Add citric acid (1.05 g) and ethylenediamine (335 μL) into deionized water (10 mL), stir evenly, transfer the solution into a high-pressure reactor, and heat at 180°C for 5 hours; after the reaction is completed, cool the reactor naturally to room temperature; obtain a brown-black transparent solution, which is then centrifuged and freeze-dried to finally obtain a brown powder.

[0020] (1.2) Thiourea (3 g) and CDs (10 mg) were added to deionized water (20 mL), dispersed evenly and freeze-dried to obtain a nanostructured precursor; the precursor was calcined, cooled, washed and vacuum-dried to obtain a CDs / C3N4 composite material.

[0021] Preferably, in step (1.2), the precursor is calcined at a temperature of 550° C. for 3 hours, and the temperature is cooled to room temperature after the calcination is completed.

[0022] Preferably, in step (1.2), the precursor is heated from room temperature to 550°C at a heating rate of 5°C min -1 .

[0023] Preferably, in step (1), the concentration of the CDs / C3N4 solution is 1-5 mg mL -1 , more preferably 2 mg mL -1 .

[0024] Preferably, in step (2), the solvent of the urease solution is acetate buffer, more preferably at pH 4.0.

[0025] Preferably, in step (2), the concentrations of the urea solutions of different concentrations are selected from different values ​​between 0-60 mM, including but not limited to the following values: 0 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, and 60 mM.

[0026] Preferably, in step (3), the concentration of the methylene blue solution is 8-12 μM, more preferably 10 μM.

[0027] Preferably, in step (4), a CDs / C3N4 / ITO electrode adsorbing methylene blue is used as a working electrode and is placed in a phosphate buffer solution (100 mM) containing ascorbic acid (100 mM), and photoelectrochemical detection of urea concentration is performed under a bias voltage of 0.05 V (vs SCE).

[0028] As another object of the present invention, the method is used to detect the urea concentration in human body fluids or environmental water.

[0029] Another object of the present invention is to use the rapid urea concentration detection method to prepare a urea concentration detection sensor, which includes a CDs / C3N4 composite material, a three-electrode system and a standard curve comparison chart.

[0030] The present invention designs a method for detecting urea concentration by using a photoelectrochemical platform for pH-regulated CDs / C3N4 surface charge adsorption of methylene blue. The method utilizes urease to catalyze the hydrolysis of urea to generate carbon dioxide and ammonia, thereby increasing the pH value of the solution, and is used to regulate the surface charge of CDs / C3N4, and gradually exhibits negative charge as the pH value increases. Methylene blue is a cationic dye and signal enhancement molecule, so CDs / C3N4 can utilize electrostatic interactions to assemble MB molecules, thereby changing the photocurrent polarity of CDs / C3N4 and enhancing the signal, and can sensitively detect urea concentration. By measuring a series of urea solutions of known concentrations, a standard curve comparison chart is obtained, and then the photocurrent value of a sample solution of unknown concentration is detected by the same method and compared with the standard curve comparison chart, the concentration of urea in the solution to be detected is obtained, and rapid detection of urea concentration is achieved. The method has the advantages of excellent selectivity, good repeatability, and the like for instant detection of urea concentration in human body fluids or the environment.

[0031] The present invention adopts the photoelectrochemical (PEC) analysis method, which has the advantages of simple instrument, low cost, high sensitivity, etc.

[0032] In the method of the present invention, since the synthesized CDs and C3N4 have band matching, the constructed heterostructure has better photoelectric properties. In addition, there are a large number of carboxyl groups and amino groups on the surface of the CDs / C3N4 material, so the charge amount carried is closely related to the pH value of the reaction system. The developed PEC sensor shows excellent analytical performance for the detection of urea, with a linear response range of 0 to 60mM. The normal content in urine and blood of normal human body is 55-388mM (9.3-23.3g / L) and 2.5-7.5mM (0.15-0.4g / L), respectively, and the urea concentration in sweat is 1.8-46mM. Therefore, the detection range of the method of the present invention meets the detection needs of urea in body fluids and can be used to detect the urea concentration of human body fluids. The biosensor prepared by the method of the present invention shows excellent selectivity, good repeatability and high stability. At the same time, the determination of urea in human body fluids and environmental water bodies was successfully achieved, indicating the reliability of the biosensor and its application prospects in clinical diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 It is a schematic diagram of the principle of the detection method of the present invention.

[0034] Figure 2 It is a comparison chart of the standard curves of different concentrations of urea in the examples. DETAILED DESCRIPTION

[0035] In order to facilitate the understanding of the present invention, the present invention will be described more comprehensively and meticulously below in conjunction with the accompanying drawings and preferred embodiments of the present invention, but the protection scope of the present invention is not limited to the following specific embodiments.

[0036] Unless otherwise defined, all the professional terms used below have the same meanings as those generally understood by those skilled in the art. The professional terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention.

[0037] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0038] In each embodiment, the method for rapid detection of urea concentration based on photoelectrochemical analysis, the drawing of the standard curve control chart, comprises the following steps:

[0039] (1) 20 μL of uniformly dispersed CDs / C3N4 solution (2 mg mL -1 ) is dropped onto an indium tin oxide glass sheet and dried for later use to form a CDs / C3N4 / ITO electrode;

[0040] (2) Add 50 μL of urease solution (containing 6 U mL urease) -1 , the solvent was acetate buffer, pH 4.0), 90 μL urease reaction buffer and 100 μL urea solution of different concentrations (0, 10, 15, 20, 30, 40, 50 mM) were added to a 96-well plate and incubated at 37 °C for 30 min;

[0041] (3) adding 10 μL of methylene blue solution (10 μM) to each of the 96-well plates of step (2), placing the CDs / C3N4 / ITO electrode obtained in step (1) into the well plate for adsorption (10 min), then taking it out, and washing the electrode three times with PBS washing solution to obtain a CDs / C3N4 / ITO electrode adsorbing methylene blue;

[0042] (4) In a three-electrode system, the CDs / C3N4 / ITO electrode adsorbed with methylene blue was used as the working electrode and placed in a phosphate buffer solution (100 mM) containing ascorbic acid (100 mM). The photoelectrochemical detection of urea concentration was performed under a bias voltage of 0.05 V (vs SCE);

[0043] (5) Draw a standard curve comparison chart based on the different concentrations of urea solutions corresponding to the different photocurrent values ​​obtained in step (4). Figure 2 .

[0044] Example 1

[0045] The principle diagram of the rapid detection method of urea concentration based on photoelectrochemical analysis in this embodiment is as follows: Figure 1 The detection method specifically comprises the following steps:

[0046] Step 1: Prepare CDs / C3N4 composite materials, urea solutions of different concentrations, urease solutions, methylene blue solutions and a three-electrode system.

[0047] Step 2: Synthesis of CDs / C3N4 composites, such as Figure 1 shown.

[0048] (1.1) Preparation of CDs

[0049] Citric acid (1.05 g) and ethylenediamine (335 μL) were added to deionized water (10 mL), stirred evenly, and then the solution was transferred to a 20 mL polytetrafluoroethylene-lined autoclave and heated at 180°C for 5 hours. After the reaction was completed, the autoclave was naturally cooled to room temperature. A brown-black transparent solution was obtained, which was then centrifuged and freeze-dried to finally obtain a brown powder (CDs).

[0050] (1.2) Preparation of CDs / C3N4

[0051] The CDs (10 mg) and thiourea (3 g) obtained in step (1.1) were added to water (20 mL), dispersed evenly, and freeze-dried to obtain a nanostructured precursor. The precursor was heated from room temperature to 550 °C and calcined at a heating rate of 5 °C min -1 , and kept at this temperature for 3 hours, then cooled to room temperature, washed and vacuum dried to obtain CDs / C3N4.

[0052] Step 3: Detect the urea content in the body fluid sample of unknown concentration.

[0053] (1) 20 μL of evenly dispersed CDs / C3N4 (2 mg mL -1 ) solution is dropped onto an indium tin oxide glass sheet and dried for later use to form a CDs / C3N4 / ITO electrode;

[0054] (2) Take a body fluid sample solution of unknown concentration, take 100 μL of the test solution, and 50 μL of urease solution (containing urease at a concentration of 6 U mL -1 , solvent was acetate buffer, pH 4.0) and 90 μL urease reaction buffer (acetate buffer, 5 mM, pH 4.0), added to a 96-well plate and incubated at 37 °C for 30 min;

[0055] (3) Repeat steps (3) and (4): add 10 μL of methylene blue solution (the concentration of methylene blue solution is 10 μM) to the 96-well plate of step (2), place the CDs / C3N4 / ITO electrode obtained in step (1) in the well plate for adsorption for ten minutes, then take it out and wash the electrode three times with PBS washing solution to obtain a CDs / C3N4 / ITO electrode adsorbing methylene blue;

[0056] (4) In a three-electrode system, the CDs / C3N4 / ITO electrode adsorbing methylene blue was used as the working electrode to perform photoelectrochemical detection of urea concentration;

[0057] The CDs / C3N4 / ITO working electrode adsorbing methylene blue is used in step (4), and the electrode is placed in a phosphate buffer solution (100 mM) containing ascorbic acid (100 mM). Photoelectrochemical detection of urea concentration is performed under the condition of applying a bias voltage of 0.05 V (vs SCE). The photocurrent reading is read, and the photocurrent value reading is compared with the standard value comparison chart to quickly know the urea concentration in the solution to be tested.

[0058] The urea concentration detected in this example is 0-60mM, of which 0-50mM is in the same linear range. This is basically consistent with the range of the existing reported wearable hydrogel patch visual monitoring of urea in body fluids (linear range: 0-40mM), proving that the biosensor prepared by the present invention exhibits excellent selectivity, good repeatability and high stability.

[0059] Example 2

[0060] The principle diagram of the rapid detection method of urea concentration based on photoelectrochemical analysis in this embodiment is as follows: Figure 1 The detection method specifically comprises the following steps:

[0061] Step 1: Prepare CDs / C3N4 composite materials, urea solutions of different concentrations, urease solutions, methylene blue solutions and a three-electrode system.

[0062] Step 2: Synthesis of CDs / C3N4 composites, such as Figure 1 shown.

[0063] (1.1) Preparation of CDs

[0064] Dissolve citric acid (1.05 g) and ethylenediamine (335 μL) in deionized water (10 mL), stir well, transfer the solution to a 20 mL Teflon-lined autoclave, and heat at 180 ° C for 5 hours. After the reaction is completed, the autoclave is naturally cooled to room temperature. A brown-black transparent solution is obtained, which is then centrifuged and freeze-dried to obtain a brown powder (CDs).

[0065] (1.2) Preparation of CDs / C3N4

[0066] The CDs (10 mg) and thiourea (3 g) obtained in step (1.1) were added to water (20 mL) and then freeze-dried to obtain the nanostructured precursors. These precursors were calcined from room temperature to 550 °C at a rate of 5 °C min -1 , and keep it at this temperature for 3 hours, then cool to room temperature, wash and dry to obtain CDs / C3N4.

[0067] Step 3: Detect the urea content in the body fluid sample of unknown concentration.

[0068] (1) 20 μL of evenly dispersed CDs / C3N4 (2 mg mL -1 ) solution is dropped onto an indium tin oxide glass sheet and dried for later use to form a CDs / C3N4 / ITO electrode;

[0069] (2) Take a body fluid sample solution of unknown concentration, take 100 μL of the solution to be tested, and 50 μL of urease solution (6 U mL -1 ) and 90 μL of urease reaction buffer (acetate buffer, 5 mM, pH 4.0) were added to a 96-well plate and incubated at 37 °C for 30 min;

[0070] (3) Repeat steps (3) and (4);

[0071] (4) The urea concentration is photoelectrochemically detected in 0.10 M PBS containing 0.10 M ascorbic acid under a bias voltage of 0.05 V using the CDs / C3N4 / ITO electrode adsorbed with methylene blue in step (4), and the photocurrent value is read. The urea concentration in the test solution can be quickly determined by comparing the photocurrent value with a standard value comparison chart.

[0072] The urea concentration detected in this example is 0-60mM, of which 0-50mM is in the same linear range. This is basically consistent with the range of the existing reported wearable hydrogel patch visual monitoring of urea in body fluids (linear range: 0-40mM), proving that the biosensor prepared by the present invention exhibits excellent selectivity, good repeatability and high stability.

[0073] Example 3

[0074] The principle diagram of the rapid detection method of urea concentration based on photoelectrochemical analysis in this embodiment is as follows: Figure 1 The detection method specifically comprises the following steps:

[0075] Step 1: Prepare CDs / C3N4 composite materials, urea solutions of different concentrations, urease solutions, methylene blue solutions and a three-electrode system.

[0076] Step 2: Synthesis of CDs / C3N4 composites, such as Figure 1 shown.

[0077] (1.1) Preparation of CDs

[0078] Dissolve citric acid (1.05 g) and ethylenediamine (335 μL) in deionized water (10 mL), stir well, transfer the solution to a 20 mL Teflon-lined autoclave, and heat at 180 ° C for 5 hours. After the reaction is completed, the autoclave is naturally cooled to room temperature. A brown-black transparent solution is obtained, which is then centrifuged and freeze-dried to obtain a brown powder (CDs).

[0079] (1.2) Preparation of CDs / C3N4

[0080] The CDs (10 mg) and thiourea (3 g) obtained in step (1.1) were added to water (20 mL) and then freeze-dried to obtain the nanostructured precursors. These precursors were calcined from room temperature to 550 °C at a rate of 5 °C min -1 , and keep it at this temperature for 3 hours, then cool to room temperature, wash and dry to obtain CDs / C3N4.

[0081] Step 3: Detect the urea content in the body fluid sample of unknown concentration.

[0082] (1): 20 μL of evenly dispersed CDs / C3N4 (2 mg mL -1 ) solution is dropped onto an indium tin oxide glass sheet and dried for later use to form a CDs / C3N4 / ITO electrode;

[0083] (2) Take a body fluid sample solution of unknown concentration, take 100 μL of the solution to be tested, and 50 μL of urease solution (6 U mL -1 ) and 90 μL urease reaction buffer (acetate buffer, 5 mM, pH 4.0) were added to a 96-well plate and incubated at 37°C for 30 min;

[0084] (3) Repeating steps (3) and (4); wherein the CDs / C3N4 / ITO electrode adsorbing methylene blue used in step (4) is used to perform photoelectrochemical detection of urea concentration in 0.10M PBS containing 0.10M ascorbic acid under a bias condition of 0.05V, and reading the photocurrent value. The photocurrent value reading is compared with the standard value comparison chart to quickly determine the urea concentration in the solution to be tested.

[0085] The urea concentration detected in this example is 0-60mM, of which 0-50mM is in the same linear range. This is basically consistent with the range of the existing reported wearable hydrogel patch visual monitoring of urea in body fluids (linear range: 0-40mM), proving that the biosensor prepared by the present invention exhibits excellent selectivity, good repeatability and high stability.

[0086] Example 4

[0087] The principle diagram of the rapid detection method of urea concentration based on photoelectrochemical analysis in this embodiment is as follows: Figure 1 The detection method specifically comprises the following steps:

[0088] Step 1: Prepare CDs / C3N4 composite materials, urea solutions of different concentrations, urease solutions, methylene blue solutions and a three-electrode system.

[0089] Step 2: Synthesis of CDs / C3N4 composites, such as Figure 1 shown.

[0090] (1.1) Preparation of CDs

[0091] Dissolve citric acid (1.05 g) and ethylenediamine (335 μL) in deionized water (10 mL), stir well, transfer the solution to a 20 mL Teflon-lined autoclave, and heat at 180 ° C for 5 hours. After the reaction is completed, the autoclave is naturally cooled to room temperature. A brown-black transparent solution is obtained, which is then centrifuged and freeze-dried to obtain a brown powder (CDs).

[0092] (1.2) Preparation of CDs / C3N4

[0093] The CDs (10 mg) and thiourea (3 g) obtained in step (1.1) were added to water (20 mL) and then freeze-dried to obtain the precursor of the nanostructure. These precursors were calcined from room temperature to 550°C at a rate of 10°C / min and kept at this temperature for 3 hours, then cooled to room temperature, washed and dried to obtain CDs / C3N4.

[0094] Step 3: Detect the urea content in the body fluid sample of unknown concentration.

[0095] (1) 20 μL of evenly dispersed CDs / C3N4 (2 mg mL -1 ) solution is dropped onto an indium tin oxide glass sheet and dried for later use to form a CDs / C3N4 / ITO electrode;

[0096] (2) Take a body fluid sample solution of unknown concentration, take 100 μL of the solution to be tested, and 50 μL of urease solution (6 U mL -1) and 90 μL urease reaction buffer (acetate buffer, 5 mM, pH 4.0) were added to a 96-well plate and incubated at 37° C. for 30 min. Steps (3) and (4) were repeated, wherein the CDs / C3N4 / ITO electrode adsorbed with methylene blue in step (4) was used to perform photoelectrochemical detection of urea concentration in 0.10 M PBS containing 0.10 M ascorbic acid under a bias condition of 0.05 V, and the photocurrent value was read. The photocurrent value was compared with the standard value comparison chart to quickly determine the urea concentration in the solution to be tested.

[0097] The urea concentration detected in this example is 0-60mM, of which 0-50mM is in the same linear range. This is basically consistent with the range of the existing reported wearable hydrogel patch visual monitoring of urea in body fluids (linear range: 0-40mM), proving that the biosensor prepared by the present invention exhibits excellent selectivity, good repeatability and high stability.

[0098] Example 5

[0099] The principle diagram of the rapid detection method of urea concentration based on photoelectrochemical analysis in this embodiment is as follows: Figure 1 The detection method specifically comprises the following steps:

[0100] Step 1: Prepare CDs / C3N4 composite materials, urea solutions of different concentrations, urease solutions, methylene blue solutions and a three-electrode system.

[0101] Step 2: Synthesis of CDs / C3N4 composites, such as Figure 1 shown.

[0102] (1.1) Preparation of CDs

[0103] Dissolve citric acid (1.05 g) and ethylenediamine (335 μL) in deionized water (10 mL), stir well, transfer the solution to a 20 mL Teflon-lined autoclave, and heat at 180 ° C for 5 hours. After the reaction is completed, the autoclave is naturally cooled to room temperature. A brown-black transparent solution is obtained, which is then centrifuged and freeze-dried to obtain a brown powder (CDs).

[0104] (1.2) Preparation of CDs / C3N4

[0105] The CDs (10 mg) and thiourea (3 g) obtained in step (1.1) were added to water (20 mL) and then freeze-dried to obtain the precursor of the nanostructure. These precursors were calcined from room temperature to 550°C at a rate of 5°C / min and kept at this temperature for 3 hours, then cooled to room temperature, washed and dried to obtain CDs / C3N4.

[0106] Step 3: Detect the urea content in the body fluid sample of unknown concentration.

[0107] (1) 20 μL of evenly dispersed CDs / C3N4 (2 mg mL -1 ) solution is dropped onto an indium tin oxide glass sheet and dried for later use to form a CDs / C3N4 / ITO electrode;

[0108] (2) Take a body fluid sample solution of unknown concentration, take 100 μL of the test solution, 50 μL of urease solution (6 U / mL) and 90 μL of urease reaction buffer (acetate buffer, 5 mM, pH 4.0) and add them to a 96-well plate, incubate at 37° C. for 30 min, and repeat steps (3) and (4), wherein the CDs / C3N4 / ITO electrode adsorbed with methylene blue in step (4) is used to perform photoelectrochemical detection of urea concentration in 10 mM PBS containing 0.10 M ascorbic acid under a bias condition of 0.05 V, read the photocurrent value, and compare the photocurrent value with the standard value comparison chart to quickly know the urea concentration in the test solution.

[0109] The urea concentration detected in this example is 0-60mM, of which 0-50mM is in the same linear range. This is basically consistent with the range of the existing reported wearable hydrogel patch visual monitoring of urea in body fluids (linear range: 0-40mM), proving that the biosensor prepared by the present invention exhibits excellent selectivity, good repeatability and high stability.

[0110] Example 6

[0111] The principle diagram of the rapid detection method of urea concentration based on photoelectrochemical analysis in this embodiment is as follows: Figure 1 The detection method specifically comprises the following steps:

[0112] Step 1: Prepare CDs / C3N4 composite materials, urea solutions of different concentrations, urease solutions, methylene blue solutions and a three-electrode system.

[0113] Step 2: Synthesis of CDs / C3N4 composites, such as Figure 1 shown.

[0114] (1.1) Preparation of CDs

[0115] Dissolve citric acid (1.05 g) and ethylenediamine (335 μL) in deionized water (10 mL), stir well, transfer the solution to a 20 mL Teflon-lined autoclave, and heat at 180 ° C for 5 hours. After the reaction is completed, the autoclave is naturally cooled to room temperature. A brown-black transparent solution is obtained, which is then centrifuged and freeze-dried to obtain a brown powder (CDs).

[0116] (1.2) Preparation of CDs / C3N4

[0117] The CDs (10 mg) and thiourea (3 g) obtained in step (1.1) were added to water (20 mL) and then freeze-dried to obtain the nanostructured precursors. These precursors were calcined from room temperature to 550 °C at a rate of 5 °C min -1 , and keep it at this temperature for 3 hours, then cool to room temperature, wash and dry to obtain CDs / C3N4.

[0118] Step 3: Detect the urea content in the body fluid sample of unknown concentration.

[0119] (1) 20 μL of evenly dispersed CDs / C3N4 (2 mg mL -1 ) solution is dropped onto an indium tin oxide glass sheet and dried for later use to form a CDs / C3N4 / ITO electrode;

[0120] (2) Take a body fluid sample solution of unknown concentration, take 100 μL of the solution to be tested, and 50 μL of urease solution (6 U mL -1 ) and 90 μL urease reaction buffer (acetate buffer, 5 mM, pH 4.0) were added to a 96-well plate and incubated at 37° C. for 30 min. Steps (3) and (4) were repeated, wherein the CDs / C3N4 / ITO electrode adsorbed with methylene blue in step (4) was used to perform photoelectrochemical detection of urea concentration in 0.10 M PBS containing 0.10 M ascorbic acid under a bias condition of 0.05 V, and the photocurrent value was read. The photocurrent value was compared with the standard value comparison chart to quickly determine the urea concentration in the solution to be tested.

[0121] The urea concentration detected in this example is 0-60mM, of which 0-50mM is in the same linear range. This is basically consistent with the range of the existing reported wearable hydrogel patch visual monitoring of urea in body fluids (linear range: 0-40mM), proving that the biosensor prepared by the present invention exhibits excellent selectivity, good repeatability and high stability.

Claims

1. A method for rapid detection of urea concentration based on photoelectrochemical analysis, characterized in that: The following steps are involved: (1) Drop the evenly dispersed CDs / C3N4 solution on an indium tin oxide glass sheet and dry it for later use to form a CDs / C3N4 / ITO electrode; (2) adding urease solution, urease reaction buffer and urea solutions of different concentrations into a 96-well plate and incubating at a certain temperature for a period of time; (3) Adding methylene blue solution to the 96-well plate of step (2), respectively, placing the CDs / C3N4 / ITO electrode obtained in step (1) into the well plate for adsorption for a period of time, then taking it out, and washing the electrode with PBS washing solution to obtain a CDs / C3N4 / ITO electrode adsorbing methylene blue; (4) In a three-electrode system, the CDs / C3N4 / ITO electrode adsorbing methylene blue was used as the working electrode to perform photoelectrochemical detection of urea concentration; (5) Drawing a standard curve comparison chart based on the different concentrations of urea solutions corresponding to the different photocurrent values ​​obtained in step (4); (6) Repeat steps (1) to (4) for a urea solution of unknown concentration to be tested to obtain a photocurrent value, and compare the value with the standard curve control chart described in step (5) to determine the urea concentration of the solution to be tested.

2. The method for rapid detection of urea concentration based on photoelectrochemical analysis according to claim 1, characterized in that: The following steps are involved: (1) Drop 20 μL of evenly dispersed CDs / C3N4 solution on an indium tin oxide glass sheet and dry it for later use to form a CDs / C3N4 / ITO electrode; (2) Add 50 µL of urease solution, 90 µL of urease reaction buffer, and 100 µL of urea solution of different concentrations into a 96-well plate and incubate at 37 °C for 30 min. (3) Add 10 µL of methylene blue solution to each of the 96-well plates in step (2), place the CDs / C3N4 / ITO electrode obtained in step (1) into the well plate for adsorption, then take it out and wash the electrode with PBS washing solution to obtain a CDs / C3N4 / ITO electrode adsorbing methylene blue; (4) In a three-electrode system, the CDs / C3N4 / ITO electrode adsorbing methylene blue was used as the working electrode to perform photoelectrochemical detection of urea concentration; (5) Drawing a standard curve comparison chart based on the different concentrations of urea solutions corresponding to the different photocurrent values ​​obtained in step (4); (6) Repeat steps (1) to (4) for a urea solution of unknown concentration to be tested to obtain a photocurrent value, and compare the value with the standard curve control chart described in step (5) to determine the urea concentration of the solution to be tested.

3. The method for rapid detection of urea concentration based on photoelectrochemical analysis according to claim 1 or 2, characterized in that: The preparation method of CDs / C3N4 composite material comprises the following steps: (1.1) Synthesis of carbon quantum dots: Add citric acid and ethylenediamine to deionized water, stir well, transfer the solution to a high-pressure reactor, and heat at 180 °C for 5 hours. After the reaction is completed, cool the reactor naturally to room temperature. A brown-black transparent solution is obtained, which is then centrifuged and freeze-dried to obtain a brown powder. (1.2) Add thiourea and CDs into deionized water, disperse them evenly and then freeze-dry them to obtain a nanostructured precursor; calcine the precursor, cool it, wash it and vacuum dry it to obtain a CDs / C3N4 composite material.

4. The method for rapid detection of urea concentration based on photoelectrochemical analysis according to claim 1 or 2, characterized in that: In step (1.2), the precursor is calcined at 550 °C for 3 hours, and then cooled to room temperature after calcination; and / or, in step (1.2), the precursor is heated from room temperature to 550 °C at a heating rate of 5 °C min -1 .

5. The method for rapid detection of urea concentration based on photoelectrochemical analysis according to claim 1 or 2, characterized in that: In step (1), the concentration of CDs / C3N4 solution is 1-5 mg mL -1 .

6. The method for rapid detection of urea concentration based on photoelectrochemical analysis according to claim 1 or 2, characterized in that: In step (2), the solvent of the urease solution is acetate buffer; and / or, in step (2), the concentrations of the urea solutions of different concentrations are selected from different values ​​between 0 and 60 mM.

7. The method for rapid detection of urea concentration based on photoelectrochemical analysis according to claim 1 or 2, characterized in that: In step (3), the concentration of the methylene blue solution is 8-12 µM.

8. The method for rapid detection of urea concentration based on photoelectrochemical analysis according to claim 1 or 2, characterized in that: In step (4), a CDs / C3N4 / ITO electrode adsorbing methylene blue is used as a working electrode and placed in a phosphate buffer solution containing ascorbic acid. Photoelectrochemical detection of urea concentration is performed under a bias voltage of 0.05 V.

9. An application of the method for rapid detection of urea concentration based on photoelectrochemical analysis according to any one of claims 1 to 8, characterized in that: The detection method is used to detect the urea concentration in human body fluids or environmental water.

10. An application of the method for rapid detection of urea concentration based on photoelectrochemical analysis according to any one of claims 1 to 8, characterized in that: A urea concentration detection sensor is prepared by using the urea concentration rapid detection method. The detection sensor comprises a CDs / C3N4 composite material, a three-electrode system and a standard curve comparison chart.

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