A rapid detection method for urea concentration based on photoelectrochemical analysis and application thereof

Abstract

A rapid detection method of urea concentration based on photoelectrochemical analysis and application thereof, the rapid detection method of urea concentration based on photoelectrochemical analysis comprises the following steps: (1) preparing a CDs / C3N4 / ITO electrode; (2) adding urease solution, urease reaction buffer and urea solution with different concentrations into a 96-well plate and incubating; (3) preparing a CDs / C3N4 / ITO electrode adsorbed with methylene blue; (4) photoelectrochemically detecting the urea concentration; (5) drawing a standard curve control diagram; and (6) detecting the concentration of urea solution with unknown concentration. The detection method is used for detecting the urea concentration in human body fluid or environmental water. The rapid detection method of urea concentration can be used to prepare a urea concentration detection sensor. The detection method has excellent selectivity, good repeatability and other advantages for instant detection of the urea concentration in human body fluid or environment.

Description

TECHNICAL FIELD

[0001] The present application relates to a rapid detection method of urea concentration and its application, in particular to a rapid detection method of urea concentration based on photoelectrochemical analysis and its application. BACKGROUND

[0002] Urea is the main component of nitrogen fertilizer, and the wastewater discharge in its production process and the farmland runoff in its use process will cause pollution of rivers, lakes and groundwater sources, become an important source of ammonia nitrogen and nitrate pollutants in water body, and make eutrophication increasingly serious, at the same time, cause groundwater pollution. For example, a large amount of nitrogen fertilizer into water body provides rich nitrogen nutrition conditions for rapid proliferation of "red tide organisms", and has become one of the main inducing factors of red tide. The occurrence of red tide destroys the marine ecosystem, and poisons or kills fish and shellfish, and endangers human health through food chain.

[0003] As an important metabolic product in human liver, the disorder of urea level in body fluid composition is closely related to many diseases such as heart and kidney failure, so the accurate detection of urea concentration in human body fluid is very important in clinical diagnosis. The traditional urea detection methods mainly include gas chromatography, calorimetry and fluorescence analysis. Although these methods are reliable, they all need high cost, large instruments and complex operation, and are not suitable for on-site monitoring. SUMMARY

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

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

[0006] (1) dropping a uniformly dispersed CDs / C3N4 solution on an indium tin oxide glass sheet, drying for standby, and constituting a CDs / C3N4 / ITO electrode;

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

[0008] (3) adding methylene blue solution into 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, taking out, washing the electrode with PBS washing solution, and obtaining a CDs / C3N4 / ITO electrode adsorbed with methylene blue;

[0009] (4) In a three-electrode system, the CDs / C3N4 / ITO electrode adsorbed with methylene blue is used as the working electrode to photoelectrochemically detect the concentration of urea;

[0010] (5) The standard curve control diagram is drawn according to the different concentrations of urea solutions corresponding to the different photocurrent values obtained in step (4);

[0011] (6) The unknown concentration of urea solution to be detected is repeated from step (1) to step (4) to obtain a photocurrent value, and the urea concentration of the solution to be detected is known by comparing the value with the standard curve control diagram described in step (5).

[0012] Preferably comprising the following steps:

[0013] (1) 20 μL of uniformly dispersed CDs / C3N4 solution is dropped on an indium tin oxide glass sheet and dried for standby, forming a CDs / C3N4 / ITO electrode;

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

[0015] (3) 10 μL of methylene blue solution is added to the 96-well plate of step (2), respectively, the CDs / C3N4 / ITO electrode obtained in step (1) is placed in the well plate for adsorption (preferably for 8-12 min, more preferably for 10 min), then taken out, and the electrode is washed with PBS washing solution three times to obtain the CDs / C3N4 / ITO electrode adsorbed with methylene blue;

[0016] (4) In a three-electrode system, the CDs / C3N4 / ITO electrode adsorbed with methylene blue is used as the working electrode to photoelectrochemically detect the concentration of urea;

[0017] (5) The standard curve control diagram is drawn according to the different concentrations of urea solutions corresponding to the different photocurrent values obtained in step (4);

[0018] (6) The unknown concentration of urea solution to be detected is repeated from step (1) to step (4) to obtain a photocurrent value, and the urea concentration of the solution to be detected is known by comparing the value with the standard curve control diagram described in step (5).

[0019] The preparation method of the CDs / C3N4 composite material is also the prior art, and the following method can also be used for preparation, comprising the following steps: (1.1) synthesis of carbon quantum dots (CDs): citric acid (1.05 g) and ethylenediamine (335 μL) are added to deionized water (10 mL), and after stirring uniformly, the solution is transferred to a high-pressure reaction kettle, and heated at 180°C for 5 hours; after the reaction is completed, the reaction kettle is naturally cooled to room temperature; a brown-black transparent solution is obtained, and then centrifuged, freeze-dried, and finally a brown powder is obtained.

[0020] (1.2) Thio urea (3 g) and CDs (10 mg) are added to deionized water (20 mL), uniformly dispersed, and then freeze-dried to obtain a nanostructured precursor; the precursor is 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 after calcination, the temperature is lowered to room temperature.

[0022] Preferably, in step (1.2), the precursor is heated from room temperature to 550°C at a 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 urea solution is an acetate buffer, more preferably with a pH of 4.0.

[0025] Preferably, in step (2), the urea solution has a concentration of 0-60 mM, and different values are selected. Including but not limited to the following values: 0 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 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), the CDs / C3N4 / ITO electrode adsorbed with methylene blue is used as the working electrode, and is placed in a phosphate buffer solution (100 mM) containing ascorbic acid (100 mM), and the photoelectrochemical detection of urea concentration is carried out under the condition of applying a bias voltage of 0.05 V (vs SCE).

[0028] Another object of the present application is that the method is used for detecting the urea concentration in human body fluids or environmental water bodies.

[0029] The application also aims to prepare a urea concentration detection sensor by using the urea concentration rapid detection method, and the detection sensor comprises CDs / C3N4 composite material, a three-electrode system and a standard curve control diagram.

[0030] The application designs a photoelectrochemical platform for urea concentration detection by adjusting the surface charge of CDs / C3N4 to adsorb methylene blue, and the method comprises the following steps: preparing a CDs / C3N4 photoelectrode; preparing a three-electrode system; preparing a standard curve control diagram; and detecting the concentration of urea in a sample solution.

[0031] The application adopts a photoelectrochemical (PEC) analysis method, and has the advantages of simple instrument, low cost, high sensitivity and the like.

[0032] In the method, the synthesized CDs and C3N4 have band matching, and the heterostructure constructed has better photoelectric performance. BRIEF DESCRIPTION OF DRAWINGS

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

[0034] Figure 2 It is a standard curve control diagram of different concentrations of urea in the embodiment. DETAILED DESCRIPTION

[0035] In order to facilitate the understanding of the present application, the following will be a more comprehensive and detailed description of the present application in conjunction with the drawings of the specification and the preferred embodiments, but the protection scope of the present application is not limited to the following specific embodiments.

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The professional terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the protection scope of the present application.

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

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

[0039] (1) 20 μL of uniformly dispersed CDs / C3N4 solution (2 mg mL -1 ) was dropped on an indium tin oxide glass sheet and dried for standby, forming a CDs / C3N4 / ITO electrode;

[0040] (2) 50 μL of urease solution (containing urease at a concentration of 6 U mL -1 , solvent is acetate buffer, pH 4.0), 90 μL of urease reaction buffer and 100 μL of 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) 10 μL of methylene blue solution (10 μM) was added to the 96-well plate of step (2), and the CDs / C3N4 / ITO electrode obtained in step (1) was placed in the well plate for adsorption (10 min) and then taken out, and the electrode was washed with PBS washing solution three times to obtain a methylene blue-adsorbed CDs / C3N4 / ITO electrode;

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

[0043] (5) The standard curve control chart was drawn according to the different photoelectric current values obtained in step (4) corresponding to different concentrations of urea solution. See 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 shown in Figure 1 The detection method specifically includes the following steps:

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

[0047] Step 2: Synthesis of CDs / C3N4 composite material, as shown in Figure 1

[0048] (1.1) Preparation of CDs

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

[0050] (1.2) Preparation of CDs / C3N4

[0051] CDs (10 mg) obtained in step (1.1) and thiourea (3 g) were added to water (20 mL), and after being uniformly dispersed, the precursor was freeze-dried. The precursor was heated from room temperature to 550°C 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 unknown concentration of body fluid sample.

[0053] (1) 20 μL of uniformly dispersed CDs / C3N4 (2 mg mL -1 ) solution was dropped on an indium tin oxide glass sheet and dried for use, forming a CDs / C3N4 / ITO electrode;

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

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

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

[0057] In step (4), use the methylene blue adsorbed CDs / C3N4 / ITO working electrode, place the electrode in a phosphate buffer solution (100 mM) containing ascorbic acid (100 mM), and perform photoelectrochemical detection of urea concentration under the condition of applying a bias of 0.05 V (vs SCE). Read the photocurrent reading, and compare the photocurrent value reading with the standard value comparison chart to quickly determine the urea concentration in the solution to be detected.

[0058] The urea concentration detected in this embodiment is 0-60 mM, of which 0-50 mM is in the same linear range. This is basically consistent with the range of the existing reported wearable hydrogel patch method for visual monitoring of urea in body fluid (linear range: 0-40 mM), which proves that the biosensor prepared in the present application 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 shown in Figure 1 The detection method specifically includes the following steps:

[0061] First step: prepare CDs / C3N4 composite material, different concentrations of urea solution, urease solution, methylene blue solution and three-electrode system.

[0062] Second step: synthesis of CDs / C3N4 composite material, as shown in Figure 1

[0063] (1.1) Preparation of CDs

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

[0065] (1.2) Preparation of CDs / C3N4

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

[0067] Third step: detecting the urea content in the unknown concentration of body fluid sample.

[0068] (1) 20 μL of uniformly dispersed CDs / C3N4(2 mg mL -1 ) solution was dropped on an indium tin oxide glass sheet and dried for use, constituting a CDs / C3N4 / ITO electrode;

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

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

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

[0072] The urea concentration detected in this example is 0-60 mM, of which 0-50 mM is in the same linear range. This is basically consistent with the range of the existing reported wearable hydrogel patch method for visual monitoring of urea in body fluids (linear range: 0-40 mM), which proves that the biosensor prepared in the present application 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 example is shown in Figure 1

[0075] ​First step: Preparation of CDs / C3N4 composite, different concentrations of urea solution, urease solution, methylene blue solution and three-electrode system.

[0076] Second step: Synthesis of CDs / C3N4 composite, as shown in Figure 1

[0077] (1.1) Preparation of CDs

[0078] Dissolve citric acid (1.05 g) and ethylenediamine (335 μL) in deionized water (10 mL), after stirring evenly, transfer the solution to a 20 mL teflon-lined autoclave, heat at 180℃ for 5 hours. After the reaction is completed, the reaction kettle is naturally cooled to room temperature. A brownish black transparent solution is obtained, then centrifuged, freeze-dried, and finally a brownish powder (CDs) is obtained.

[0079] (1.2) Preparation of CDs / C3N4

[0080] Add CDs (10 mg) obtained in step (1.1) and thiourea (3 g) to water (20 mL), then freeze-dry to obtain nanostructured precursors. These precursors are calcined from room temperature to 550℃ at a rate of 5℃ min -1 , and kept at this temperature for 3 hours, then reduced to room temperature, washed and dried to obtain CDs / C3N4.

[0081] Third step: Detection of urea content in unknown concentration of body fluid sample.

[0082] (1): Drop 20 μL of uniformly dispersed CDs / C3N4 (2 mg mL -1 ) solution on an indium tin oxide glass sheet, dry for use, and constitute a CDs / C3N4 / ITO electrode;

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

[0084] (3) Repeat steps (3) and (4); wherein the CDs / C3N4 / ITO electrode adsorbed with methylene blue is used in step (4), and the urea concentration is photoelectrochemically detected in 0.10 M PBS containing 0.10 M ascorbic acid under the condition of applying a bias of 0.05 V, and the photocurrent value reading is read, and by comparing the photocurrent value reading with the standard value comparison chart, the urea concentration in the sample to be tested can be quickly determined.

[0085] ​The embodiment detects the urea concentration of 0-60 mM, of which 0-50 mM is in the same linear range. This is basically consistent with the existing reported wearable hydrogel patch method for visual monitoring of urea in body fluid (linear range: 0-40 mM), which proves that the biosensor prepared by the application 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 the embodiment is shown in Figure 1 The detection method specifically includes the following steps:

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

[0089] Step 2: Synthesis of CDs / C3N4 composite material, as shown in Figure 1

[0090] (1.1) Preparation of CDs

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

[0092] (1.2) Preparation of CDs / C3N4

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

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

[0095] (1) Drop 20 μL of uniformly dispersed CDs / C3N4 (2 mg mL -1 ) solution on an indium tin oxide glass sheet and dry for use, to form a CDs / C3N4 / ITO electrode;

[0096] (2) Take an unknown concentration of body fluid sample solution, take 100 μL of the sample solution to be tested, 50 μL of urease solution (6 U mL -1 ​) and 90 μL urease reaction buffer (acetate buffer, 5 mM, pH 4.0) were added into a 96-well plate, incubated at 37 °C for 30 min, and steps (3) and (4) were repeated, in which the urea concentration was photoelectrochemically detected by using the CDs / C3N4 / ITO electrode adsorbed with methylene blue in 0.10 M PBS containing 0.10 M ascorbic acid under a bias of 0.05 V, and the photocurrent value reading was read, which was compared with the standard value comparison chart, so as to quickly know the urea concentration in the solution to be detected.

[0097] The urea concentration in this embodiment was detected in the range of 0-60 mM, and 0-50 mM was in the same linear range. This is basically consistent with the existing reported wearable hydrogel patch method for visual monitoring of urea in body fluid (linear range: 0-40 mM), which proves that the biosensor prepared by the present application 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 shown in Figure 1 The detection method specifically includes the following steps:

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

[0101] Step 2: Synthesis of CDs / C3N4 composite material, as shown in Figure 1

[0102] (1.1) Preparation of CDs

[0103] Citric acid (1.05 g) and ethylenediamine (335 μL) were dissolved in deionized water (10 mL), and after stirring, the solution was transferred to a 20 mL teflon-lined autoclave, heated at 180 °C for 5 hours. After the reaction was completed, the reaction kettle was naturally cooled to room temperature. A brown-black transparent solution was obtained, then centrifuged, freeze-dried, and finally a brown powder (CDs) was obtained.

[0104] (1.2) Preparation of CDs / C3N4

[0105] CDs (10 mg) obtained in step (1.1) and thiourea (3 g) were added to water (20 mL), then freeze-dried to obtain nanostructured precursors. 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 reduced to room temperature, washed and dried to obtain CDs / C3N4. ​

[0106] Third step: detecting the urea content in the unknown concentration of body fluid sample.

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

[0108] (2) An unknown concentration of body fluid sample solution was taken, 100 μL of the sample solution, 50 μL of urease solution (6 U / mL) and 90 μL of urease reaction buffer (acetate buffer, 5 mM, pH 4.0) were added into a 96-well plate, and incubated at 37 °C for 30 min, and then steps (3) and (4) were repeated, wherein the urea concentration was photoelectrochemically detected by using the methylene blue adsorbed CDs / C3N4 / ITO electrode in 10 mM PBS containing 0.10 M ascorbic acid under the bias of 0.05 V, and the photocurrent value reading was read, and the photocurrent value reading was compared with the standard value, so that the urea concentration in the sample solution could be quickly determined.

[0109] The urea concentration detected in this example was 0-60 mM, and 0-50 mM was in the same linear range. This is basically consistent with the range of the existing reported wearable hydrogel patch method for visual monitoring of urea in body fluid (linear range: 0-40 mM), which proves that the biosensor prepared in the present application 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 example is shown in Figure 1 The detection method specifically includes the following steps:

[0112] First step: preparing CDs / C3N4 composite material, different concentrations of urea solution, urease solution, methylene blue solution and three-electrode system.

[0113] Second step: synthesis of CDs / C3N4 composite material, as shown in Figure 1

[0114] (1.1) Preparation of CDs

[0115] Citric acid (1.05 g) and ethylenediamine (335 μL) were dissolved in deionized water (10 mL), and after stirring uniformly, the solution was transferred to a 20 mL teflon-lined autoclave, and heated at 180 °C for 5 hours. After the reaction was completed, the reaction kettle was naturally cooled to room temperature. A brownish black transparent solution was obtained, which was then centrifuged, freeze-dried, and finally a brownish powder (CDs) was obtained.​​

[0116] (1.2) Preparation of CDs / C3N4

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

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

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

[0120] (2) Take an unknown concentration of body fluid sample solution, take 100 μL of the sample to be tested, 50 μL of urease solution (6 U mL -1 ) and 90 μL of urease reaction buffer (acetate buffer, 5 mM, pH 4.0) into a 96-well plate, incubate at 37°C for 30 min, and then repeat steps (3) and (4), wherein the CDs / C3N4 / ITO electrode adsorbed with methylene blue is used in step (4), and the urea concentration is photoelectrochemically detected under the condition of applying a bias voltage of 0.05 V in 0.10 M PBS containing 0.10 M ascorbic acid, and the photocurrent value reading is read, and the photocurrent value reading is compared with the standard value comparison chart, so that the urea concentration in the sample to be tested can be quickly determined.

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

Claims

1. A rapid detection method of urea concentration based on photoelectrochemical analysis, characterized in that, It comprises the following steps: (1) Drop the uniformly dispersed CDs / C3N4 solution on the indium tin oxide glass sheet, dry for standby, and constitute the CDs / C3N4 / ITO electrode; (2) Add urease solution, urease reaction buffer and different concentrations of urea solution into the 96-well plate, and incubate at a certain temperature for a period of time; (3) Add methylene blue solution into the 96-well plate of step (2) respectively, put the CDs / C3N4 / ITO electrode obtained in step (1) into the well plate for adsorption, take it out after a period of time, wash the electrode with PBS washing solution, and obtain the CDs / C3N4 / ITO electrode adsorbed with methylene blue; (4) In a three-electrode system, take the CDs / C3N4 / ITO electrode adsorbed with methylene blue as the working electrode, and photoelectrochemically detect the urea concentration; (5) Draw a standard curve control diagram according to the different photoelectric current values obtained in step (4) corresponding to different concentrations of urea solution respectively; (6) Repeat steps (1) to (4) for the unknown concentration urea solution to be detected, obtain a photoelectric current value, and compare it with the standard curve control diagram in step (5) to know the urea concentration of the solution. The preparation method of the CDs / C3N4 composite material comprises the following steps: (1.1) Synthesis of carbon quantum dots: add citric acid and ethylenediamine into deionized water, stir uniformly, then transfer the solution into a high-pressure reaction kettle, heat at 180℃ for 5 hours; after the reaction is completed, naturally cool the reaction kettle to room temperature; obtain a brown-black transparent solution, then centrifuge, freeze-dry, and finally obtain a brown powder; (1.2) Add thiourea and CDs into deionized water, disperse uniformly, then freeze-dry to obtain a nanostructured precursor; calcine the precursor, cool down, wash and vacuum dry to obtain the CDs / C3N4 composite material.

2. The rapid detection method of urea concentration based on photoelectrochemical analysis according to claim 1, characterized in that, It comprises the following steps: (1) Drop 20 µL of uniformly dispersed CDs / C3N4 solution on the indium tin oxide glass sheet, dry for standby, and constitute the CDs / C3N4 / ITO electrode; (2) Add 50 µL of urease solution, 90 µL of urease reaction buffer and 100 µL of different concentrations of urea solution into the 96-well plate, and incubate at 37℃ for 30 min; (3) Add 10 µL of methylene blue solution into the 96-well plate of step (2) respectively, put the CDs / C3N4 / ITO electrode obtained in step (1) into the well plate for adsorption, take it out after a period of time, wash the electrode with PBS washing solution, and obtain the CDs / C3N4 / ITO electrode adsorbed with methylene blue; (4) In a three-electrode system, take the CDs / C3N4 / ITO electrode adsorbed with methylene blue as the working electrode, and photoelectrochemically detect the urea concentration; (5) Draw a standard curve control diagram according to the different photoelectric current values obtained in step (4) corresponding to different concentrations of urea solution respectively; (6) Repeat steps (1) to (4) for the unknown concentration urea solution to be detected, obtain a photoelectric current value, and compare it with the standard curve control diagram in step (5) to know the urea concentration of the solution. 3.The rapid detection method 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 a temperature of 550 °C for 3 hours, and then cooled to room temperature; and / or, in step (1.2), the precursor is heated from room temperature to 550 °C at a rate of 5 °C min -1 . 4.The rapid detection method of urea concentration based on photoelectrochemical analysis according to claim 1 or 2, characterized in that, In step (1), the concentration of the CDs / C3N4 solution is 1-5 mg mL -1 .

5. The rapid detection method 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 concentration of the urea solution is selected from a value between 0-60 mM. 6.The rapid detection method 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. 7.The rapid detection method of urea concentration based on photoelectrochemical analysis according to claim 1 or 2, characterized in that, In step (4), the CDs / C3N4 / ITO electrode adsorbing methylene blue is used as a working electrode, and is placed in a phosphate buffer solution containing ascorbic acid, and the photoelectrochemical detection of urea concentration is carried out under the condition of applying a bias voltage of 0.05 V.

8. An application of the method for rapid detection of urea concentration based on photoelectrochemical analysis according to any one of claims 1 to 7, characterized in that: The detection method is used for detecting the urea concentration in human body fluid or environmental water.

9. Use of a rapid test method for the determination of the concentration of urea based on photoelectrochemical analysis according to any one of claims 1 to 7, characterized in that, A urea concentration detection sensor is prepared by using the rapid urea concentration detection method, and the detection sensor comprises a CDs / C3N4 composite material, a three-electrode system and a standard curve control diagram.

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