A method for detecting ascorbic acid in urine based on nitrogen and boron co-doped fluorescent carbon dots

Abstract

The application discloses a method for detecting ascorbic acid in urine based on nitrogen and boron co-doped fluorescent carbon dots, and belongs to the field of analysis and detection. The carbon dots are prepared by taking citric acid, ethylenediamine and boric acid as precursors, and the fluorescent quantum yield of the carbon dots reaches 84.5%. The high iodine oxide oxidizes the surface functional groups of the carbon dots to generate non-fluorescent groups, so that the fluorescence of the carbon dots is quenched. The ascorbic acid preferentially reacts with the high iodine oxide in an oxidation-reduction reaction, thereby inhibiting the fluorescence quenching effect of the high iodine oxide, and the inhibition degree is related to the concentration of the ascorbic acid. Based on this, a relationship curve between the concentration of the ascorbic acid and the degree of inhibition of fluorescence quenching is constructed, so that the quantitative detection of the concentration of the ascorbic acid in urine is realized, and the detection limit is as low as 0.01 nmol / L. The application first uses an "inhibition" type fluorescence sensor to quantitatively detect the ascorbic acid, and is simple, safe, efficient and suitable for routine analysis.

Description

TECHNICAL FIELD

[0001] The application relates to a method for detecting ascorbic acid in urine based on nitrogen-boron co-doped fluorescent carbon dots, and belongs to the field of analysis and detection. BACKGROUND

[0002] Ascorbic acid (AA), also known as vitamin C, is an important micronutrient and antioxidant, and is an indispensable water-soluble vitamin in the human body. It can scavenge free radicals and is related to key processes of human metabolism, including immune function, cell differentiation and iron absorption, and plays an important role in cell physiological activities and homeostasis of the human body. For example, AA can provide protection against free radical damage and diseases such as Parkinson's syndrome and various cancers. However, a lack of AA in the human body can cause skin bruising, gum bleeding, muscle weakness, joint swelling and edema, while excessive AA intake can cause urinary stones, diarrhea and stomach cramps. Therefore, it is essential to develop a method that is efficient, sensitive, specific and can detect trace amounts of AA.

[0003] In recent years, various analysis methods for detecting AA have been developed. Common AA analysis methods include high-performance liquid chromatography, enzyme-linked immunoassay, electrochemical method, colorimetric method, capillary electrophoresis method, etc. These methods have high precision and reliability, but have problems such as poor selectivity, high toxicity, low sensitivity and complex operation. Therefore, there is an urgent need to find a simpler, faster and more sensitive AA detection method. SUMMARY

[0004] Technical problem:

[0005] The various analysis methods for detecting ascorbic acid developed at present mainly include high-performance liquid chromatography, electrochemical method, chemiluminescence method, etc. These methods have problems such as poor selectivity, high toxicity, low sensitivity and complex operation. At the same time, there are few reports on the use of fluorescent sensing technology to detect ascorbic acid in urine, and the sensitivity and detection limit are not superior to traditional methods.

[0006] Technical scheme:

[0007] A method for detecting ascorbic acid in urine based on nitrogen-boron co-doped fluorescent carbon dots, comprising the following steps:

[0008] (1) Synthesizing a nitrogen-boron co-doped fluorescent carbon dot solution by one-step hydrothermal method with citric acid, ethylenediamine and boric acid as precursors, purifying and diluting the solution for subsequent detection;

[0009] (2) prepare a standard sodium periodate solution and a series of ascorbic acid standard solutions with known concentrations; mix the ascorbic acid standard solutions with the urine sample, then add the sodium periodate solution and the buffer solution, and finally add the purified and diluted nitrogen-boron co-doped fluorescent carbon dot solution, mix, incubate, obtain the corresponding test sample liquid, and perform fluorescence spectrum detection;

[0010] (3) the fluorescence intensity peak values after adding ascorbic acid with a concentration of 0 and not 0 are F1 and F respectively, and the inhibition degree of fluorescence quenching is calculated as (F-F1) / F1; a linear quantitative detection model is constructed by taking the inhibition degree of fluorescence quenching and the corresponding ascorbic acid concentration as variables.

[0011] (4) refer to step (2) to add the sodium periodate solution and the buffer solution to the urine sample to be detected, and finally add the purified and diluted nitrogen-boron co-doped fluorescent carbon dot solution, mix, incubate, obtain the corresponding test sample liquid; perform fluorescence spectrum detection; and obtain the concentration of ascorbic acid in the urine sample to be detected according to the linear model in step (3).

[0012] In an embodiment of the present application, in step (1), the mass ratio of citric acid to boric acid is 4:1.

[0013] In an embodiment of the present application, in step (1), the amount of ethylenediamine relative to citric acid is 1 mL / g.

[0014] In an embodiment of the present application, in step (1), the volume ratio of ethylenediamine to water in the hydrothermal method is 1:9.

[0015] In an embodiment of the present application, in step (1), the hydrothermal method is under the condition of 200℃ for 6h.

[0016] In an embodiment of the present application, in step (1), the purification step of the carbon dots is as follows: centrifuge the crude carbon dot solution synthesized at 10000 rpm for 8 min, then filter the unreacted particles through a microporous filter membrane with a pore size of 0.22 μM, and finally dialyze the carbon dots through a dialysis membrane with a molecular weight cut-off of 1000 Da for 24h to obtain the final carbon dots.

[0017] In an embodiment of the present application, in step (1), the dilution multiple of the purified carbon dots is 5000 times.

[0018] In an embodiment of the present application, in step (2), the fluorescence spectrum detection is performed under the following conditions: the fluorescence spectrum is measured by a fluorescence spectrometer, the excitation slit and emission slit widths of the spectrometer are 2 nm and 2.5 nm respectively, and the integration time is 0.1 s; the excitation wavelength of the fluorescence spectrometer is 349 nm, the emission wavelength range is 380 nm-600 nm, and the step is 1 nm.

[0019] In one embodiment of the present invention, the incubation time in step (2) is 30 minutes.

[0020] In one embodiment of the present invention, the buffer solution in step (2) is phosphate buffer (PBS) with a concentration of 10 mM and pH = 7.4.

[0021] In one embodiment of the present invention, the volume ratio of urine, ascorbic acid, sodium periodate, phosphate buffer and carbon dot solution in step (2) is 1:1:1:1:1.

[0022] In one embodiment of the present invention, the concentration of the standard sodium periodate solution in step (2) is 1 mmol / L.

[0023] In one embodiment of the present invention, the linear relationship described in step (3) is:

[0024] (F-F1) / F1=1.38573c(AA)-0.05805, where F1 and F represent the fluorescence intensity of the system after adding ascorbic acid at a concentration of 0 or not, respectively, and c(AA) represents the concentration of ascorbic acid.

[0025] The present invention also provides the application of the method in the field of biomedical detection.

[0026] Beneficial effects:

[0027] Existing methods for detecting ascorbic acid (AA) using carbon dots as probes are relatively limited and rarely applied to trace amounts of AA. Furthermore, there are no reports of indirect detection of trace AA using IO4- as an intermediate. This invention utilizes a "suppressed" fluorescence sensor for the detection of ascorbic acid in urine. The carbon dots are spherical or near-spherical in shape and contain abundant functional groups on their surface, improving their water solubility and affinity to IO4-. - The ability to combine;

[0028] This invention synthesizes nitrogen-boron co-doped carbon dots (N,B-CDs) using citric acid as the carbon source, ethylenediamine as the nitrogen source, and boric acid as the boron source. - It has a significant fluorescence quenching effect, and the addition of ascorbic acid inhibits IO4. - The fluorescence is quenched, thus enabling the quantitative detection of ascorbic acid in urine. This method is simple, rapid, and safe, making it suitable for routine analysis. In this invention, IO4... - It will oxidize the functional groups on the surface of carbon dots and produce non-fluorescent groups, causing fluorescence quenching, while ascorbic acid and IO4 - Redox reactions occur preferentially, thus inhibiting IO4. -Different concentrations of ascorbic acid exhibit different inhibitory effects on fluorescence quenching. Based on this, an "inhibition" fluorescence sensor can detect trace amounts of ascorbic acid in urine.

[0029] The linear range for detecting ascorbic acid in this invention is 0-2 nmol / L, with a detection limit of 0.01 nmol / L. The recovery rate for urine samples is 96%-102%. Compared to other fluorescence detection methods, such as those based on Fe... 3+ This invention is the first to use an "off-on" type fluorescence sensor with IO4. - The development of an "inhibition" fluorescent sensor using this medium enables trace detection of ascorbic acid with an extremely low detection limit, which is of great significance to the biomedical field. Attached Figure Description

[0030] Figure 1 This is a flowchart for detecting ascorbic acid in urine using nitrogen-boron co-doped carbon dots.

[0031] Figure 2 The results are the fluorescence response test results before and after the addition of sodium periodate and ascorbic acid in Example 2.

[0032] Figure 3 This illustrates the effect of different components on carbon dot fluorescence in Example 2.

[0033] Figure 4 The fluorescence spectra are shown for different concentrations of ascorbic acid added to the system in Example 3.

[0034] Figure 5 The curve showing the relationship between the degree of inhibition of fluorescence quenching and the concentration of ascorbic acid in Example 3 is shown.

[0035] Figure 6 The curves show the linear fit between the degree of inhibition of fluorescence quenching in Example 3 and the concentration range of ascorbic acid (0-2 nM).

[0036] Figure 7 This is a graph showing the test results for the selectivity of ascorbic acid in Example 4.

[0037] Figure 8 The image shows the test results for the anti-interference ability of ascorbic acid in Example 5.

[0038] Figure 9 The graph shows the test results of the effect of periodate concentration in Example 6.

[0039] Figure 10 The graph shows the test results of the effect of pH on the detection effect of phosphate buffer solution in Example 7.

[0040] Figure 11 The graph shows the test results of the effect of reaction time on the detection of ascorbic acid in Example 8. Detailed Implementation

[0041] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be regarded as limiting the scope of the present invention.

[0042] Example 1: Preparation of Carbon Dot (CDs) Solution

[0043] Weigh 2.0 g of citric acid and 0.5 g of boric acid into a beaker, then add 2 ml of anhydrous ethylenediamine and 18 ml of deionized water sequentially. Sonicate for 5 min to ensure complete dissolution, then react at 200 °C for 6 h. Centrifuge the synthesized crude carbon dot solution at 10,000 rpm for 8 min, collect the supernatant, and then filter through a 0.22 μM microporous membrane to remove unreacted particles. Finally, dialysis the carbon dots using a dialysis membrane with a molecular weight cutoff of 1000 Da for 24 h to obtain the final carbon dots. Subsequent experiments used carbon dot solutions based on this method.

[0044] Example 2: Preliminary Investigation of IO4 - Fluorescence response of ascorbic acid before and after the addition of carbon dots

[0045] Take three test tubes and label them 1, 2, and 3. Add ascorbic acid (0.4 ml, 5 mM) standard solution to test tube 3. Then add equal volumes of IO4- to test tubes 2 and 3. - The standard solution (10 mM) was prepared, and finally, equal volumes of carbon dot solution were added to three test tubes, and the volume was brought to 4 ml with deionized water. After incubation for a period of time, fluorescence detection was performed, and the results are as follows. Figure 2 As shown, IO4 - It has a significant quenching effect on the fluorescence of carbon dots; when ascorbic acid is present, IO4... - Fluorescence quenching was significantly suppressed. To rule out the individual effect of ascorbic acid on carbon dots, fluorescence spectra of ascorbic acid and carbon dots coexisting were supplemented, and the test results are as follows. Figure 3 As shown, the fluorescence intensity of the carbon dots remained essentially unchanged before and after the addition of ascorbic acid, with only the fluorescence intensity of IO4 increasing. - In its presence, the fluorescence of carbon dots is quenched, and when ascorbic acid, IO4 - When the three elements (ascorbic acid, carbon dots, and iodine) coexist, fluorescence quenching is inhibited, further demonstrating that the addition of ascorbic acid can inhibit IO4. - This method is feasible for quenching fluorescence.

[0046] Example 3: Constructing a linear determination model for ascorbic acid

[0047] Preparation of sample solutions: carbon dot solution (obtained by diluting the carbon dot solution obtained in Example 1 by 5000 times), phosphate buffer (pH = 7.4), IO4 - Standard solution (1 mM), urine sample, and ascorbic acid standard solutions with concentrations of 0 (blank control), 0.1 nM, 0.3 nM, 0.5 nM, 0.7 nM, 1 nM, 1.5 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, and 8 nM;

[0048] First, add a series of ascorbic acid standard solutions of various concentrations and urine (0.4 ml each) to a test tube. Then, add 0.4 ml of 1 mM IO4 solution sequentially. - The solution and 0.4 ml phosphate buffer (PBS, 10 mM, pH = 7.4) were mixed, and then 0.4 ml carbon dot solution was added. The mixture was then brought to a final volume of 4 ml with deionized water and allowed to stand for 30 min to obtain urine test sample solutions with different ascorbic acid concentrations. Fluorescence spectroscopy was then performed to detect the samples.

[0049] The fluorescence spectrum of the determination system was measured under the following conditions: excitation wavelength of 349 nm, emission wavelength scanning range of 380-600 nm, scanning every 1 nm, and slit width of 2 nm / 2.5 nm (excitation slit / emission slit). A fluorescence intensity peak at 441 nm was obtained. A sample solution with 0% ascorbic acid was added for fluorescence spectroscopy detection, yielding a fluorescence intensity peak F1 at 441 nm. Sample solutions with other concentrations of ascorbic acid were used to obtain fluorescence intensity peaks F. The degree of inhibition of fluorescence quenching was recorded as (F-F1) / F1. Figure 4 As shown.

[0050] Plot the relationship between the degree of inhibition of fluorescence quenching by ascorbic acid in the sample solution and the concentration of ascorbic acid, such as... Figure 5 As shown, the linear fitting curves for the degree of fluorescence quenching inhibition and ascorbic acid concentration are as follows: Figure 6 ,from Figure 5 , Figure 6 It can be seen that when the ascorbic acid concentration is 0-2 nM, the degree of inhibition of fluorescence quenching is linearly related to the ascorbic acid concentration, and the linear equation is (F-F1) / F1=1.38573c(AA)-0.05805, with a correlation coefficient of R. 2 =0.99327, detection limit is 0.01nM.

[0051] Example 4 investigates the selectivity of nitrogen-boron co-doped carbon dots in detecting ascorbic acid in urine.

[0052] Referring to Example 3, 17 substances commonly found in urine (Ca) 2+ Cl - Cu2+ K + Mg 2+ NH4 + SO4 2- Zn 2+ P2O7 4- L-cysteine, glutathione, glutamine, creatinine, allantoin, urea, uric acid, and glucose were used as interfering agents to study the selectivity for detecting ascorbic acid in urine. The concentrations of ascorbic acid and the interfering agents were both 1 mM, IO4. - The concentration was fixed at 1 mM, and the blank control was the result without the addition of ascorbic acid (i.e., only IO4). - The fluorescence intensity of the system (quenching effect on carbon dot fluorescence). Detection results are as follows: Figure 7 As shown.

[0053] As can be seen from the image, only after ascorbic acid is added to the system does IO4 increase. - The quenching effect on carbon dot fluorescence was suppressed, while other substances could not suppress fluorescence quenching at all.

[0054] Example 5 investigates the anti-interference ability of nitrogen-boron co-doped carbon dots in detecting ascorbic acid in urine.

[0055] Referring to Examples 3 and 4, the same 17 substances were used as interfering agents to study the anti-interference effect of detecting ascorbic acid in urine. Ascorbic acid and the interfering agents were mixed, and the test sample solution was prepared according to the procedure in Example 3. The concentrations of both ascorbic acid and the interfering agents were 1 mM, IO4. - The concentration was fixed at 1 mM, and the blank control was the result without the addition of ascorbic acid (i.e., only IO4). - The fluorescence intensity of the system (quenching effect on carbon dot fluorescence). Detection results are as follows: Figure 8 As shown.

[0056] As can be seen from the image, when ascorbic acid and the interferon are added simultaneously, IO4 - The quenching effect on carbon dot fluorescence was still suppressed, and the system maintained a high fluorescence intensity.

[0057] Combined with Examples 4 and 5, it was demonstrated that the "suppression" type fluorescence sensor can detect ascorbic acid in urine with high selectivity and sensitivity, further illustrating the feasibility and accuracy of this detection scheme.

[0058] Example 6: Investigating the detection of ascorbic acid in IO4 - Effect of concentration

[0059] Referring to Example 3, carbon dot solution was added to IO4 solutions of different concentrations (0.2 μM, 0.5 μM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM). -In the standard solution, the measured results are as follows: Figure 9 As shown, when IO4 - At a concentration of 1 mM, the fluorescence quenching rate already exceeds 60%. Therefore, when detecting ascorbic acid, IO4+ should be used. - The concentration was fixed at 1 mM.

[0060] Example 7 Investigates the effect of phosphate buffer on the detection of ascorbic acid

[0061] Referring to step (2) in Example 3, phosphate buffer solutions with different pH values ​​(5-10) were added to ascorbic acid and IO4. - Finally, carbon dot solution is added to the mixed solution to fix IO4. - The concentration was 1 mM, and the measured results were as follows: Figure 10 As shown, ascorbic acid exhibits the most ideal inhibition of fluorescence quenching when the pH of the buffer solution is 6. Considering that the detection environment is urine, the pH of the buffer solution is fixed at 7.4 when detecting ascorbic acid in urine.

[0062] Example 8 investigates the effect of reaction time on the detection of ascorbic acid.

[0063] Referring to Example 3, the carbon dot solution was added to IO4. - In the standard solution, the fluorescence spectrum of the system was recorded every minute, and the results are as follows: Figure 11 As shown, when the reaction time of the system reaches 30 min, the fluorescence intensity tends to stabilize and there is basically no significant change. Therefore, before performing fluorescence testing on ascorbic acid, all samples are incubated for 30 min.

[0064] In conjunction with Examples 6, 7, and 8, when detecting ascorbic acid in urine, IO4 was used... - The concentration was fixed at 1 mM, the pH of the phosphate buffer was fixed at 7.4, and the incubation time of the reaction solution was controlled at 30 min. Under these conditions, fluorescence spectroscopy was finally performed.

[0065] Example 9: Detection of ascorbic acid in urine environment

[0066] Referring to Example 3, ascorbic acid at concentrations of 1, 1.5, and 2 nM was measured. The results are shown in Table 1. The results are satisfactory, indicating that this method is accurate and feasible, and can be applied to the detection of ascorbic acid in the biomedical field.

[0067] Table 1. Results of ascorbic acid detection in urine.

[0068] Spiked concentration (nM) Detection concentration (nM) Recovery (%) Relative standard deviation (%), n = 3 1.0 1.02 102.0 1.68 1.5 1.49 99.3 2.62 2.0 1.92 96.0 3.16

[0069] Example 10: Comparison of detection limits for ascorbic acid using different methods

[0070] The detection limit obtained by the method in Example 3 of this invention was compared with other fluorescence detection methods, and the results are shown in Table 2. The data in Table 2 clearly show that the carbon dot assay synthesized using citric acid, ethylenediamine, and boric acid as raw materials can achieve an extremely low detection limit for ascorbic acid, demonstrating that the method of this invention has high sensitivity for detecting ascorbic acid.

[0071] Table 2 Comparison of detection limits of different methods

[0072]

[0073] Among them, [1] Li,

[0074] [2]Hao,Y.,et al.,The on-off-on Fluorescence Sensor of Hollow CarbonDots for Detecting Hg(2+)and Ascorbic Acid.J Fluoresc, 2023.33(2):p.459-469.

[0075] [3]Ye, S., et al., Fluorine-Nitrogen-Codoped Carbon Dots as FluorescentSwitch Probes for Selective Fe(III)and Ascorbic Acid Sensing in LivingCells. Molecules, 2022.27(19).

[0076] [4] Preethi, M., et al., Potato starch derived N-doped carbon quantumdots as a fluorescent sensing tool for ascorbic acid. Journal of Photochemistry and Photobiology A: Chemistry, 2022.431.

[0077] [5]Hu,Y.,et al.,A rapid and sensitive turn-on fluorescent probe forascorbic acid detection based on carbon dots–MnO2 nanocomposites.AnalyticalMethods,2017.9(38):p.5653-5658.

[0078] [6]Xu,H.-B.,et al.,Preparation of biomass-waste-derived carbon dotsfrom apricot shell for highly sensitive and selective detection of ascorbicacid.Chinese Journal of Analytical Chemistry,2022.50(12).

[0079] [7]Zhang,Y.,et al.,Carbon Quantum Dots as Fluorescence Turn-Off-OnProbe for Detecting Fe(3+)and Ascorbic Acid.J Nanosci Nanotechnol,2020.20(6):p.3340-3347.

[0080] [8]Lin,Y.,et al.,An“on-off-on”fluorescent system based on themicrowave-assisted preparation of copper-functionalized carbon quantum dotsfor sensitive detection of ascorbic acid.Optical Materials,2021.115.

[0081] [9] Huang, D., et al., A Continuously Tunable Full-Color EmissionNitrogen-Doped Carbon Dots and for Ultrasensitive and Highly Selective Detection of Ascorbic Acid. Nanomaterials (Basel), 2022.12(4).

[0082] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method for detecting ascorbic acid in urine based on nitrogen-boron co-doped fluorescent carbon dots, characterized in that, Includes the following steps: (1) A nitrogen-boron co-doped fluorescent carbon dot solution was synthesized by a one-step hydrothermal method using citric acid, ethylenediamine and boric acid as precursors. The solution was then purified and diluted for subsequent detection. (2) Prepare a standard sodium periodate solution and a series of ascorbic acid standard solutions of known concentrations; The ascorbic acid standard solution was mixed with the urine sample, then sodium periodate solution and buffer were added, and finally purified and diluted nitrogen-boron co-doped fluorescent carbon dot solution was added. The mixture was mixed, incubated, and the corresponding test sample solution was obtained for fluorescence spectroscopy detection. (3) The fluorescence intensity peaks after adding ascorbic acid with a concentration of 0 and a non-zero concentration are F1 and F, respectively. The degree of inhibition of fluorescence quenching is calculated as (F-F1) / F1. A linear quantitative detection model is constructed based on the degree of inhibition of fluorescence quenching and the corresponding ascorbic acid concentration. (4) Add sodium periodate solution and buffer solution to the urine sample to be tested as in step (2), and finally add purified and diluted nitrogen-boron co-doped fluorescent carbon dot solution, mix well, incubate, and obtain the corresponding sample solution to be tested; perform fluorescence spectroscopy detection; and obtain the concentration of ascorbic acid in the urine sample to be tested according to the linear model in step (3).

2. The method according to claim 1, characterized in that, In step (1), the mass ratio of citric acid to boric acid is 4:

1.

3. The method according to claim 1, characterized in that, In step (1), the amount of ethylenediamine relative to citric acid is 1 mL / g.

4. The method according to claim 1, characterized in that, In step (1), the volume ratio of ethylenediamine to water in the hydrothermal method is 1:

9.

5. The method according to claim 1, characterized in that, In step (1), the purification steps of carbon dots are as follows: the synthesized crude carbon dot solution is centrifuged at 10,000 rpm for 8 min, then filtered through a 0.22 μM microporous membrane to remove unreacted particles, and finally purified by dialysis with a dialysis membrane with a molecular weight cutoff of 1000 Da for 24 h to obtain the final carbon dots.

6. The method according to claim 1, characterized in that, In step (1), the carbon dots are diluted by 5000 times after purification.

7. The method according to claim 1, characterized in that, The conditions for fluorescence spectroscopy detection in step (2) are as follows: the fluorescence spectrum is measured using a fluorescence spectrometer, the excitation slit width and emission slit width of the spectrometer are 2nm and 2.5nm respectively, the integration time is 0.1s; the excitation wavelength of the fluorescence spectrometer is 349nm, the emission wavelength range is 380nm-600nm, and the step size is 1nm.

8. The method according to claim 1, characterized in that, In step (2), the volume ratio of urine, ascorbic acid, sodium periodate, phosphate buffer, and carbon dot solution is 1:1:1:1:

1.

9. The method according to claim 1, characterized in that, The concentration of the standard sodium periodate solution in step (2) is 1 mmol / L.

10. The method according to any one of claims 1-9, characterized in that, The incubation time mentioned in step (2) is 30 minutes.

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