Method for detecting urea in urine based on pH sensitive carbon dots and urease

Abstract

The invention discloses a method for detecting urea in urine based on pH sensitive carbon dots and urease, and belongs to the technical field of analysis and detection. According to the method for detecting the urea in the urine based on the pH-sensitive carbon dots and the urease, the urea in the urine is detected on the basis that the pH-sensitive carbon is synthesized through a hydrothermal method by taking isoprenaline hydrochloride as a precursor, the detection sensitivity is high, and the specificity and the anti-interference performance are high; the method is simple, can realize high-efficiency detection of urea in a complex liquid environment of urine, is simple, has strong operability, and has wide application prospects in the field of medical detection.

Description

Technical Field

[0001] This invention relates to a method for detecting urea in urine based on pH-sensitive carbon dots and urease, belonging to the field of analytical detection technology. Background Technology

[0002] Urea, a nitrogenous end product of protein metabolism in the human body, is mainly synthesized by the liver and excreted through the kidneys. Its concentration level is one of the core indicators for clinically assessing renal excretory function and overall nitrogen metabolism status. As diabetes progresses, persistent hyperglycemia easily induces renal microvascular complications (i.e., diabetic nephropathy), leading to gradual damage to kidney function. Urea is widely present in human body fluids such as blood, urine, and saliva, and the concentration of urinary urea is significantly higher than that of blood urea. Compared to invasive blood tests, non-invasive urine sampling combined with urea testing can more directly and safely reflect changes in the body's physiological state. Therefore, abnormal fluctuations in urinary urea concentration have become a key biomarker for characterizing the progression of diabetic nephropathy and the degree of renal function impairment. Its accurate detection is of great significance for the early screening of diabetic nephropathy, dynamic assessment of the disease, and adjustment of clinical intervention plans.

[0003] Currently, the main techniques used in clinical urea quantitative analysis include capillary electrophoresis, electrochemical methods, and high-performance liquid chromatography (HPLC). While capillary electrophoresis offers advantages such as high detection sensitivity and low detection limits, it suffers from drawbacks including expensive equipment, complex sample pretreatment procedures, and demanding operational requirements, making it unsuitable for primary healthcare settings and rapid testing needs. Electrochemical sensors, with their low cost and fast response speed, are suitable for rapid on-site testing, but they are susceptible to interference from environmental factors such as temperature, humidity, and cross-contamination, resulting in insufficient detection stability and accuracy. HPLC possesses strong anti-interference capabilities and can adapt to various testing scenarios, but its time-consuming process and high equipment and operating costs also prevent it from meeting the needs of rapid clinical testing and large-scale screening.

[0004] With the rapid development of nanotechnology, the innovative applications of novel nanomaterials in analytical chemistry have attracted widespread attention from the academic community. Carbon dots (CDs), as a type of zero-dimensional carbon-based nanomaterial, possess excellent photochemical stability, unique optical properties, and good biocompatibility. They have achieved breakthrough applications in cutting-edge fields such as biosensing, in vivo imaging, and molecular labeling, providing new technical ideas for constructing highly sensitive, highly stable, rapid, and convenient detection methods.

[0005] Given the current situation, existing urea detection technologies suffer from problems such as complex operation, high cost, and insufficient stability, which have not yet been effectively resolved. However, the unique advantages of carbon dot materials provide a feasible path for optimizing urea detection technology. Therefore, developing a rapid, simple, and accurate urea content determination method based on carbon dots is of significant scientific value and clinical application potential for overcoming existing technological deficiencies, meeting the detection needs of clinical and primary healthcare settings, and promoting the development of early screening technology for diabetic nephropathy. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the purpose of this invention is to provide a method for detecting urea in urine based on pH-sensitive carbon dots and urease. This method uses isoproterenol hydrochloride as a precursor to synthesize a pH-sensitive carbon dot via a hydrothermal method. Based on the mechanisms that the fluorescence intensity of this carbon dot changes with pH and that urease hydrolyzes urea, leading to an increase in pH, this invention applies it to the detection of urea in urine, enabling simple, efficient, and sensitive quantitative and qualitative detection of urea in urine without pretreatment.

[0007] To achieve the above objectives, the following technical solution is provided: The present invention also provides a method for detecting urea in urine based on pH-sensitive carbon dots and urease. The method involves mixing pretreated urine with urease and pH-sensitive carbon dot solutions, incubating at room temperature, and then performing fluorescence spectroscopy detection. The fluorescence intensity value at 443±5 nm is substituted into a standard curve model to calculate the urea content in the urine to be tested.

[0008] In one embodiment, the pretreated urine refers to urine obtained by centrifugation, obtaining the supernatant, and filtering.

[0009] In one embodiment, the centrifugation conditions are: 10,000~15,000 rpm for 10~15 min.

[0010] In one embodiment, the filtration is performed using a 0.22 μM microporous membrane.

[0011] In one embodiment, the pretreated urine may be diluted by an appropriate factor, such as 50 to 200 times, before being used for subsequent measurements.

[0012] In one embodiment, the concentration of the urease is 0.1~0.2 mg / mL.

[0013] In one embodiment, the incubation time is 1 to 3 hours.

[0014] In one embodiment, the conditions for fluorescence spectroscopy detection are as follows: the excitation slit width of the spectrometer is 2 nm, the emission slit width is 2 nm, and the integration time is 0.1 s; the excitation wavelength of the fluorescence spectrometer is 350 nm, the emission wavelength range is 360~600 nm, and the step size is 1~2 nm.

[0015] In one embodiment, the standard curve model is constructed by using a series of urea standard solutions of different concentrations as standards for measurement, and constructing a standard curve model with the fluorescence intensity at 443±5 nm as the ordinate and the urea concentration as the abscissa.

[0016] In one embodiment, the pH-sensitive carbon dot solution is generated by a hydrothermal reaction of isoproterenol hydrochloride and water.

[0017] In one embodiment, the pH-sensitive carbon dot solution is prepared as follows: Isoproterenol hydrochloride was dissolved in water and then transferred to a reaction vessel for reaction. After the reaction, the solution was centrifuged, filtered, and purified by dialysis to obtain a pH-sensitive carbon dot solution.

[0018] In one embodiment, the isoproterenol hydrochloride is fully dissolved in water to obtain a solution concentration of 20-50 mg / mL.

[0019] In one embodiment, the reaction is carried out at a temperature of 150-200°C for 8-12 hours.

[0020] In one embodiment, the centrifugation parameters are 8000~15000 rpm and the time is 10~30 min.

[0021] In one embodiment, the filtration uses a 0.2~0.25 μM microporous membrane.

[0022] In one embodiment, the dialysis is performed using a dialysis membrane with a molecular weight cutoff of 500-3500 Da for dialysis purification for 12-24 h.

[0023] In one embodiment, the pH-sensitive carbon dot solution needs to be diluted as needed during actual testing, typically by a factor of 20 to 40.

[0024] In one embodiment, the pH-sensitive carbon dot solution is synthesized by hydrothermal reaction of 0.47g of isoproterenol hydrochloride in 10mL of deionized water, and then diluted 40 times with deionized water.

[0025] In one embodiment, each 4 mL detection system contains 1 mL of pretreated urine, 0.2 mL of urease solution, and 100 μL of pH-sensitive carbon dot solution, with the remainder being water; the concentration of the urease solution is 0.1~0.2 mg / mL.

[0026] Beneficial effects: (1) The present invention uses isoproterenol hydrochloride as a precursor to synthesize carbon dots, and uses them as a fluorescent sensor to realize the quantitative detection of urea. This method is simple and fast to operate, has a wide linear range, can detect urea of ​​0~0.1mM, has high detection sensitivity, and a detection limit as low as 0.017mM. (2) In this invention, the ratio of the absorption peaks of the fluorescence excitation spectrum of the pH-sensitive carbon point and the ultraviolet-visible absorption spectrum after mixing with urea changes, and it has good selectivity for urea; in addition, the pH-sensitive carbon point has strong anti-interference ability and can also detect urea in the complex liquid environment of urine, which is of great significance in the field of medical testing. Attached Figure Description

[0027] Figure 1 The following are performance characterization diagrams of the pH-sensitive carbon dots (CDs) prepared in Example 1: (a) Fluorescence excitation and emission spectra of the CDs aqueous solution; (b) Fluorescence emission spectra of the CDs at different pH (2-11) (excitation wavelength 350 nm); (c) Peak intensity variation of the CDs with pH. Figure 2 The following are data graphs of the standard curve model constructed in Example 2: (a) is a graph showing the relationship between the peak fluorescence intensity of the urea-urease-carbon dot (urea concentration of 0.1-200 μM) mixed solution and the urea concentration; (b) is a graph showing the relationship between the fluorescence intensity of the urea solution and the urea concentration range of 0.1-200 μM; (c) is a graph showing the linear fitting curve of the peak fluorescence intensity and the urea concentration range of 0.1-100 μM. Figure 3 The graph shows the specificity and anti-interference data of pH-sensitive carbon dots (CDs) in Example 3; (a) shows specificity; (b) shows anti-interference. Figure 4 Performance characterization of CDs prepared for Comparative Example 1; (a) Fluorescence emission spectra at different pH (2-11); (b) Peak intensity variation of CDs with pH; Figure 5 Performance characterization of CDs prepared in Comparative Example 2; (a) Fluorescence emission spectra at different pH (1-10); (b) Peak intensity variation of CDs with pH; Figure 6 The color changes of carbon dots, carbon dot A and carbon dot B of isoproterenol hydrochloride in the pH range of 1–11. Detailed Implementation

[0028] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. The specific embodiments described below further illustrate the present invention.

[0029] The source of raw materials involved in this invention: Isoproterenol hydrochloride was purchased from Shanghai Sinopharm Chemical Reagent Co., Ltd., China. Urease was purchased from Shanghai Yeyuan Co., Ltd., China (1.1u / mg solid).

[0030] Example 1 A method for preparing pH-sensitive carbon dots includes the following: Weigh 0.47 g of isoproterenol hydrochloride into a beaker, add 10 mL of deionized water, sonicate for 10 min, and after complete dissolution, transfer to a 25 mL polytetrafluoroethylene liner. React at 180 °C for 8 h. After the reaction is complete, centrifuge the reaction system at 10,000 rpm for 30 min, filter it using a 0.22 μM microporous membrane, and then dialyze it using a dialysis membrane with a molecular weight cutoff of 1000 Da for 24 h to obtain the carbon dot CDs mother liquor.

[0031] Performance Characterization The fluorescence excitation and emission spectra of CDs are as follows: Figure 1 As shown in (a).

[0032] Using the carbon dot mother liquor obtained above, 0.1 mL of carbon dot mother liquor and 3.9 mL of PBS buffer (concentration of 0.1 M) with different pH values ​​(2-11) were mixed and then incubated at 25 °C for 1 h to obtain carbon dot solutions under different pH conditions, and fluorescence spectroscopy was performed.

[0033] The results are as follows Figure 1 As shown, Figure 1 (b) Emission spectra of carbon dots (CDs) mixed with buffer solutions of different pH values ​​at an excitation wavelength of 350 nm. As can be seen from the figure, the fluorescence intensity of CDs decreases significantly with increasing pH value of the buffer solution; especially in the pH range of 4 to 8 (which coincides with the pH range of human urine), the fluorescence response intensity quenching amplitude reaches about 17.8 times. Figure 1 (c) shows the relationship between the fluorescence peak wavelength of CDs and pH value. The two show an excellent linear relationship in the pH range of 4 to 7, with a correlation coefficient R² = 0.99.

[0034] Example 2 A method for detecting urea in urine based on pH-sensitive carbon dots and urease, the method comprising the following steps: (1) Construction of the standard curve model Preparation of sample solutions: carbon dot solution (prepared by diluting the carbon dot stock solution obtained in Example 1 by 40 times), urease solution (0.1 mg / mL), and urea standard solutions with concentrations of 0 μmol / L (blank control), 10 μmol / L, 20 μmol / L, 30 μmol / L, 40 μmol / L, 50 μmol / L, 60 μmol / L, 70 μmol / L, 80 μmol / L, 90 μmol / L, 100 μmol / L, 110 μmol / L, 120 μmol / L, 130 μmol / L, 140 μmol / L, 150 μmol / L, 160 μmol / L, 170 μmol / L, 180 μmol / L, 190 μmol / L, and 200 μmol / L. 0.2 mL of urease solution and 1 mL of urea standard solutions of different concentrations were mixed, and then 100 μL of carbon dot solution was added. The mixture was then brought to a final volume of 4 mL with deionized water and incubated at 25 °C for 2 h to obtain spiked carbon dot solutions of different urea concentrations. Fluorescence spectroscopy was then performed. The scanning conditions were: excitation wavelength 350 nm, emission wavelength scanning range 360 ​​~ 600 nm, scanning every 1 nm, and slit width set to 2 nm / 2 nm (excitation slit / emission slit). The resulting fluorescence emission spectra were... Figure 2 a) The relationship curve between fluorescence intensity peak and urea concentration, as shown in the figure. Figure 2 As shown in b-2c, when the urea concentration is 0~0.1 mmol / L, the fitted curves of the fluorescence intensity peak and the urea concentration show that the fluorescence intensity peak at 443 nm has a linear relationship with the urea concentration. The linear equation is F = 123138.48 - 963.89C, and the correlation coefficient is R. 2 =0.991, detection limit is 0.0017 mmol / L; (2) Determination of urea in the sample to be tested Mix 0.2 mL of urease solution with 1 mL of the sample to be tested, add 100 μL of carbon dot solution, and finally dilute to 4 mL with deionized water. Then incubate at 25 °C for 2 h and perform fluorescence spectroscopy detection. Fluorescence spectrum: Scanning conditions: excitation wavelength is 350 nm, emission wavelength scanning range is 360 ~ 600 nm, scan once every 1 nm, slit width is set to 2 nm / 2 nm (excitation slit / emission slit), take the fluorescence intensity peak at 443 nm, and calculate the urea content in the sample to be tested according to the standard curve model constructed in step (1).

[0035] Example 3: Selectivity and Anti-interference Capability Referring to the determination method in Example 2, different types of inorganic substances (KBr, NaCl, NaNO3, CaCl2, BaSO4, acetone, urea) and amino acids (arginine, glycine, tryptophan, serine) with a concentration of 1 mmol / L were mixed with urease solution (10 mg / L), and then mixed with carbon dot solution (prepared by diluting the carbon dot mother liquor obtained in Example 1 by 40 times). After incubation at 25°C for 2 h, fluorescence spectroscopy was performed.

[0036] The results are as follows Figure 3 As shown, F0 and F represent the fluorescence intensity before and after the addition of the analyte, respectively. All fluorescence detections were performed under the same conditions. The results indicate that the fluorescence response intensity of carbon dots to other inorganic substances and amino acids is relatively low. Except for urea, the interference substances in urine, when mixed with urease, have little effect on the fluorescence intensity of CDs. However, urea can be hydrolyzed by urease, increasing the solution pH, which significantly reduces the fluorescence intensity of CDs. The results show that the impact of CDs on urea recognition is within an acceptable range, indicating that the system has good anti-interference ability.

[0037] Example 4 Two urine samples from healthy individuals were pretreated: 8 mL of clear urine was centrifuged at 10,000 rpm for 15 min, and the supernatant was filtered through a 0.22 μM microporous membrane and diluted 100 times to obtain the pretreated urine solution. The pretreated urine solution (0.04 mL) was mixed with 1 mL of urea standard solutions of 20, 40, and 50 μmol / L, respectively, and then 200 μL of urease solution of 0.1 mg / mL was added and mixed thoroughly. 0.1 mL of carbon dot solution (concentration: 40 times diluted with the mother liquor) was mixed with urease-urine pretreatment solution containing different concentrations of urea, and then the volume was adjusted to 4 mL with deionized water. After incubation at 25°C for 2 h, carbon dot solutions with different urea in the urine sample environment were obtained and fluorescence spectrometry was performed. The fluorescence intensity peak at 443 nm was then substituted into the linear relationship obtained in Example 2 for calculation. The detection results are shown in Table 1.

[0038] Table 1 Spike Recovery Rate

[0039] Comparative Example 1 Weigh 0.18 g of diaminoterephthalic acid and 1 ml of ethylenediamine into a beaker, add 10 mL of deionized water, sonicate for 10 min, and after complete dissolution, transfer to a 25 mL polytetrafluoroethylene liner. React at 180 °C for 8 h. After the reaction is complete, centrifuge the reaction system at 10,000 rpm for 30 min, filter it using a 0.22 μM microporous membrane, and then dialyze it using a dialysis membrane with a molecular weight cutoff of 1000 Da for 12 h to obtain carbon dot solution A.

[0040] Using the carbon dot solution A obtained above, following the same method as in Example 1, 0.1 mL of carbon dot solution A and 3.9 mL of PBS buffer (concentration of 0.1 M) with different pH values ​​were mixed, and then incubated at 25°C for 1 h to obtain carbon dot solutions with different pH values, and fluorescence spectroscopy was performed.

[0041] The results are as follows Figure 4 As shown, this indicates that the carbon point has a generally low sensitivity to pH, especially in the pH range of 4-8 (which is consistent with the pH range of human urine), where its fluorescence response intensity only increases by 1.5 times.

[0042] Comparative Example 2 Weigh 0.15 g of methionine and 0.19 g of citric acid into a beaker, add 10 mL of deionized water, sonicate for 10 min, and after complete dissolution, transfer to a 25 mL polytetrafluoroethylene liner. React at 180 °C for 10 h. After the reaction is complete, centrifuge the reaction system at 10000 rpm for 30 min, filter it using a 0.22 μM microporous membrane, and then dialyze it using a dialysis membrane with a molecular weight cutoff of 1000 Da for 10 h to obtain carbon dot solution B.

[0043] Using the carbon dot solution obtained above, following the same method as in Example 1, 0.1 mL of carbon dot solution B and 3.9 mL of PBS buffer (concentration of 0.1 M) with different pH values ​​were mixed, and then incubated at 25 °C for 1 h to obtain carbon dot solutions with different pH values, and fluorescence spectroscopy was performed.

[0044] The results are as follows Figure 5 As shown, this indicates that the fluorescence intensity of this carbon spot changes very little within the pH range of 4-10.

[0045] Figure 6 The graph shows the color changes of carbon dots (carbon dot A and carbon dot B) of isoproterenol hydrochloride within the pH range of 1–11. As can be seen from the graph, the color of the isoproterenol hydrochloride carbon dot system gradually changes from pale yellow to dark brown as the pH increases, while the color of carbon dots A and B shows almost no significant change. The results indicate that the isoproterenol hydrochloride carbon dots are highly sensitive to pH changes, which is directly visible to the naked eye.

[0046] Comparative Example 3 The results of comparing the carbon dot detection of urea with currently reported methods are shown in Table 2.

[0047] Table 2. Method for carbon dot detection of urea

[0048] As shown in Table 2, compared with existing carbon dot detection methods for urea, the method of the present invention has a lower detection limit and a more sensitive detection response, based on the single precursor and simplified preparation process. Furthermore, the color of the system of isoproterenol hydrochloride carbon dots gradually changes from light yellow to dark brown as the pH value increases, which can be directly observed with the naked eye and accurately used for qualitative and quantitative detection.

[0049] [1] Yin W, Zhang Y, Gu J, Wang T, Ma C, Zhu C, Li L, Yang Z, Zhu T,Chen G. Urea detection in milk by urease-assisted pH-sensitive carbon dots.Appl Opt. 2021 Nov 20;60(33):10421-10428. doi: 10.1364 / AO.437787. [2] Jia An, Yongqin Hu, Da Yang, Yaqin Han, Jiajing Zhang, Yufei Liu, pH-induced highly sensitive fluorescence detection of urea and urease based on carbon dots-based nanohybrids, Spectrochimica Acta Part A: Molecular andBiomolecular Spectroscopy, Volume 269, 2022, 120705, ISSN 1386-1425, https: / / doi.org / 10.1016 / j.saa.2021.120705. [3] Yuyang Bei, Kamile Arkin, Yuxin Zheng, Xuesong Ma, Jie Zhao, Huimin Jin, Qingkun Shang, Construction of a ratiometric fluorescent probe for visual detection of urea in human urine based on carbon dots prepared from Toona sinensis leaves and 5-carboxyfluorescein,Analytica Chimica Acta,Volume 1240, 2023,340733, ISSN 0003-2670, https: / / doi.org / 10.1016 / j.aca.2022.340733. The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. A method for detecting urea in urine based on pH-sensitive carbon dots and urease, characterized in that, The method involves mixing pretreated urine with urease and pH-sensitive carbon dot solution, incubating at room temperature, and then performing fluorescence spectroscopy detection. The fluorescence intensity value at 443±5 nm is substituted into the standard curve model to calculate the urea content in the urine to be tested. The pH-sensitive carbon dot solution is generated by hydrothermal reaction of isoproterenol hydrochloride and water.

2. The method according to claim 1, characterized in that, The concentration of the urease is 0.1~0.2 mg / mL.

3. The method according to claim 1, characterized in that, The incubation time is 1 to 3 hours.

4. The method according to claim 1, characterized in that, The conditions for fluorescence spectroscopy detection are as follows: the excitation slit width of the spectrometer is 2 nm, the emission slit width is 2 nm, and the integration time is 0.1 s; the excitation wavelength of the fluorescence spectrometer is 350 nm, the emission wavelength range is 360~600 nm, and the step size is 1~2 nm.

5. The method according to claim 1, characterized in that, The pH-sensitive carbon dot solution is prepared as follows: Isoproterenol hydrochloride was dissolved in water and then transferred to a reaction vessel for reaction. After the reaction, the solution was centrifuged, filtered, and purified by dialysis to obtain a pH-sensitive carbon dot solution.

6. The method according to claim 5, characterized in that, The isoproterenol hydrochloride is fully dissolved in water to obtain a solution concentration of 20-50 mg / mL.

7. The method according to claim 5, characterized in that, The reaction is carried out at a temperature of 150-200℃ for 8-12 hours.

8. The method according to claim 5, characterized in that, The centrifugation parameters are 8000~15000 rpm and the time is 10~30 min.

9. The method according to claim 5, characterized in that, The filtration process uses a 0.2~0.25μM microporous membrane.

10. The method according to claim 5, characterized in that, The dialysis was performed using a dialysis membrane with a molecular weight cutoff of 500-3500 Da for dialysis purification for 12-24 h.

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