A triple-emission carbon quantum dot, a preparation method thereof and application thereof in rapid detection of uric acid

Abstract

The application discloses a kind of triple emission carbon quantum dots and preparation method and application in rapid detection of uric acid, the carbon quantum dots are prepared using the following method: (1) p-phenylenediamine, malic acid and gadolinium nitrate are added to solvent, so that all raw materials are dissolved to obtain precursor solution;(2) hydrothermal reaction is carried out, after reaction is finished, remove large particle impurities and small molecule impurities, dry to obtain the carbon quantum dot powder;Wherein the molar ratio of p-phenylenediamine, malic acid and gadolinium nitrate is 1~3:1:1~4.The carbon quantum dots of the application have three independent fluorescence emission peaks, construct three-channel ratio sensing system, can specifically identify uric acid and occur selective fluorescence response, good anti-interference, high detection sensitivity, detection limit is as low as 0.054 μM, effectively solve the defects that existing single emission CDs uric acid detection sensitivity is insufficient, easily interfered, simultaneously significantly improve the sensitivity and accuracy of uric acid quantitative detection.

Description

Technical Field

[0001] This invention relates to a carbon quantum dot, its preparation method and application, and particularly to a triple-emission carbon quantum dot, its preparation method and its application in rapid uric acid detection. Background Technology

[0002] Uric acid (UA, 2,6,8-trihydroxypurine), as the end product of purine metabolism in the human body, cannot be further oxidized and decomposed due to the lack of uricase in the human body. Therefore, its concentration homeostasis in body fluids is crucial for human health. Under normal physiological conditions, the concentration of uric acid in human serum is maintained between 120.0 and 460.0 μM, and the excretion concentration in urine is between 1.4 and 4.4 mM. Abnormal fluctuations in this concentration are directly related to the occurrence and development of various diseases—high uric acid levels easily lead to gout, hyperuricemia, atherosclerosis, and arthritis, and are also closely related to cardiovascular diseases, Parkinson's disease, Alzheimer's disease, kidney damage, and metabolic syndrome; while low uric acid levels may damage the body's antioxidant system and induce infertility related to oxidative stress. Therefore, uric acid, as a key biomarker, requires rapid, accurate, and convenient detection in biological samples, which is of significant practical importance for clinical diagnosis, disease monitoring, and prognostic assessment.

[0003] In recent years, various reliable uric acid (UA) analysis strategies have emerged, including surface-enhanced Raman scattering (SERS), enzyme-linked immunosorbent assay (ELISA), high-performance liquid chromatography (HPLC), electrochemical methods, and chemiluminescence methods. While these methods can achieve accurate uric acid detection, they suffer from drawbacks such as cumbersome sample pretreatment, long detection cycles, the need for specialized operators, and expensive precision instruments, limiting their application in convenient, visualized, and real-time detection. Fortunately, fluorescence detection technology based on nanoprobes effectively overcomes these shortcomings due to its advantages of simple operation, rapid response, high sensitivity, low cost, and potential for visualized detection.

[0004] Fluorescence detection technology, with its advantages of simple operation, rapid response, high sensitivity, low cost, and the ability to achieve visual detection, has become a research hotspot in the field of uric acid point-of-care testing. Among them, fluorescence detection systems based on nanoprobes effectively overcome the shortcomings of traditional detection methods. Carbon quantum dots (CDs), as a novel class of fluorescent carbon nanomaterials with a particle size of less than 10 nm, have outstanding characteristics such as excellent photostability, tunable luminescence properties, low cytotoxicity, ease of synthesis, and good biocompatibility. They have been widely used in the field of biosensing and have become ideal materials for constructing fluorescent uric acid detection probes, providing high-quality sensing materials for the development of visual uric acid test strips. However, most of the CDs-based fluorescent probes currently used for uric acid detection are constructed based on single-emission CDs probes. These single-signal systems rely solely on the fluorescence intensity change of a single emission peak for detection. They are inherently susceptible to interference from matrix background, environmental fluctuations, and instrument instability. These factors significantly reduce the detection sensitivity and reliability in practical sample analysis and limit their application in visualized real-time detection. UA test strips based on single-emission carbon dots can only determine UA concentration through the intensity of a single fluorescence point, making them vulnerable to external interference, resulting in large visual discrimination errors and low quantitative detection accuracy, failing to meet the practical needs of precise detection. To address the inherent limitations of single-emission probes, ratiometric fluorescent probes (RFPs) have emerged. These probes achieve quantitative detection through the ratio of fluorescence intensities of two or more emission peaks, utilizing an inherent self-calibration mechanism to counteract external interference, significantly improving detection stability and accuracy, and laying the technological foundation for the development of highly accurate visualized test strips.

[0005] Currently, ratiometric fluorescent probes used for uric acid detection are mainly limited to systems constructed from dual-emission carbon dots (CDs), with a limited number of related studies. Furthermore, dual-emission systems can only provide one fluorescence intensity ratio, resulting in limited self-calibration capabilities and failing to accurately detect complex biological samples. Recently, the emergence of triple-emission carbon dots (CDs) has provided new prospects for the development of ratiometric fluorescent sensing and visual test strips. These CDs possess three independent fluorescence emission peaks and can achieve multiple self-calibrations through multiple fluorescence intensity ratios, more effectively offsetting matrix interference, environmental fluctuations, and instrument errors. This further improves the accuracy, stability, and anti-interference capabilities of the detection, offering unique advantages in the detection of complex biological samples and the preparation of visual test strips. However, to date, there have been no reports on the construction of uric acid-responsive ratiometric fluorescent probes based on triple-emission carbon dots, nor any research on visual test strips prepared based on this novel triple-emission ratiometric probe. Therefore, developing a uric acid-responsive ratiometric probe based on triple-emission carbon dots and a matching visual test strip is of great significance for improving the sensitivity, accuracy, and reliability of uric acid detection. Summary of the Invention

[0006] Objectives of the Invention: The first objective of this invention is to provide a triple-emission carbon quantum dot that improves the sensitivity and accuracy of quantitative uric acid detection; the second objective of this invention is to provide a method for preparing the triple-emission carbon quantum dot; and the third objective of this invention is to provide applications of the triple-emission carbon quantum dot.

[0007] Technical solution: The preparation method of triple-emission carbon quantum dots (TCDs) of the present invention includes the following steps:

[0008] (1) Add p-phenylenediamine, malic acid and gadolinium nitrate to a solvent to dissolve all raw materials to obtain a precursor solution;

[0009] (2) A hydrothermal reaction is carried out. After the reaction is completed, large particulate impurities and small molecule impurities are removed, and the carbon quantum dot powder is dried to obtain the powder. The molar ratio of p-phenylenediamine (MPD), malic acid and gadolinium nitrate is 1~3:1:1~4.

[0010] Preferably, the molar ratio of p-phenylenediamine, malic acid, and gadolinium nitrate is 1~2:1:1.

[0011] Preferably, the hydrothermal reaction is carried out at a temperature of 180°C for 12 hours.

[0012] Preferably, the solvent is a mixture of ethanol and water.

[0013] Triple-emission carbon quantum dots prepared by any of the methods of this invention.

[0014] The triple-emission carbon quantum dot has three distinct emission peaks at 346, 417, and 511 nm.

[0015] Preferably, the excitation wavelength of the triple-emission carbon quantum dot is 300 nm.

[0016] The test strip of the present invention contains the triple-emission carbon quantum dots.

[0017] The application of the triple-emission carbon quantum dots or the test strip described in this invention in the rapid detection of uric acid.

[0018] Preferably, the application method is as follows: a TCDs solution is prepared using a buffer solution, then different concentrations of UA are added to the prepared TCDs solution. After the reaction, the fluorescence intensity of the mixed solution at 346, 417, and 511 nm under 300 nm excitation is recorded; according to (F 1-0 +F 2-0 +F 3-0 The fluorescence quenching efficiency is ) / (F1+F2+F3), where F 1-0 F 2-0 F 3-0F1, F2, and F3 represent the initial fluorescence intensities of TCDs at wavelengths of 346, 417, and 511 nm before the addition of uric acid, respectively. F1, F2, and F3 represent the fluorescence intensities at the corresponding wavelengths after the addition of uric acid. A linear curve y = 0.0196x + 0.9083 was obtained by fitting the fluorescence intensity as y and the UA concentration as x. The detection limit of uric acid is 0.054 µM.

[0019] Preferably, the application method is as follows: under 365 nm ultraviolet light irradiation, when the uric acid concentration varies in the range of 0.0~500.0 μM, the TCDs solution exhibits a distinct color gradient from bright cyan to royal blue. The image signal is converted into RGB values ​​using the ColorColl App on a smartphone, and the blue / green channel ratio (B / G) is used as a quantitative indicator. The linear equation obtained is B / G = 0.0048[UA] + 1.0623, the correlation coefficient R² = 0.9941, and the detection limit is 2.20 μM.

[0020] Mechanism of invention: The TCDs of this invention can generate three independent fluorescence emission peaks (at 346, 417, and 511 nm) at an excitation wavelength of 300 nm, constructing a three-channel ratiometric sensing system that can specifically recognize uric acid and exhibit a selective fluorescence response—after the action of uric acid, the fluorescence intensity of the three peaks of the TCDs changes differentially, and by calculating (F... 1-0 +F 2-0 +F 3-0 ) / (F1+F2+F3)(F 1-0 F1 and F2 represent the fluorescence intensity of the emission peak at 346 nm before and after the addition of uric acid, respectively; F 2-0 F1 and F2 represent the fluorescence intensity of the emission peak at 417 nm before and after the addition of uric acid, respectively; F2 and F3 represent the fluorescence intensity of the emission peak at 417 nm before and after the addition of uric acid. 3-0 F1 and F2 represent the fluorescence intensity of the emission peak at 511 nm before and after the addition of uric acid, respectively. A self-calibration mechanism is used to offset instrument fluctuations and matrix interference, achieving highly accurate quantitative detection. Based on this, utilizing the concentration-dependent fluorescence color change from bright cyan to blue associated with uric acid, TCDs are prepared into test strips. Using a smartphone color recognition device, a portable visual detection system has been developed, enabling rapid, portable, and visual detection of uric acid in human urine.

[0021] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) The carbon quantum dots of the present invention have three independent fluorescence emission peaks, and a three-channel ratio sensing system is constructed. It can specifically identify uric acid and generate selective fluorescence response. It has good anti-interference ability, high detection sensitivity, and a detection limit as low as 0.054 μM. It effectively solves the defects of insufficient sensitivity and easy interference of existing single emission CDs for uric acid detection, and significantly improves the sensitivity and accuracy of quantitative uric acid detection. (2) The preparation method of the carbon quantum dots of the present invention is simple and low cost. (3) The carbon quantum dots of the present invention are used to detect uric acid. The detection method is simple. Under ultraviolet light irradiation, after the sample is added, it can show a visually distinguishable fluorescence color change with the increase of uric acid concentration. Combined with the color recognition device of a smartphone, it can realize portable visual rapid detection of uric acid in urine. The detection operation is fast and convenient, and a good recovery rate (96.1%~106.2%) was obtained with a relative standard deviation of less than 3.07%. Attached Figure Description

[0022] Figure 1 The FTIR spectrum of the TCDs prepared in Example 1;

[0023] Figure 2 (A) TEM image of TCDs prepared in Example 1; (B) histogram of particle size distribution of TCDs prepared in Example 1;

[0024] Figure 3 (A) UV-Vis absorption, fluorescence excitation and emission spectra of TCDs prepared in Example 1; (B) Fluorescence spectra of TCDs at different excitation wavelengths, with inserted images showing TCDs in aqueous solution under natural light (left) and UV irradiation (right); (C) (a) Quinine sulfate and (b) UV absorption intensity-fluorescence peak area integral plots of TCDs.

[0025] Figure 4 (A) Fluorescence decay curves of TCDs before and after the introduction of UA; (B) UV-Vis absorption spectrum of UA, fluorescence excitation spectrum and emission spectrum of TCDs.

[0026] Figure 5 Fluorescence emission spectra of carbon dots (TCDs) prepared by MPD with different molar ratios of malic acid, gadolinium nitrate hexahydrate (Gd(NO3)3·6H2O), and MPD at different excitation wavelengths λex: (A) 1:1:1, (B) 1:1:2, (C) 1:1:3, (D) 1:1:4, (E) 2:1:1, (F) 2:1:3, (G) 3:1:1, and (H) 3:1:2;

[0027] Figure 6 (A) TCDs concentration; (B) and (C) Effect of reaction time on UA ​​detection;

[0028] Figure 7 (A) Fluorescence spectra of TCDs solutions containing different concentrations of UA; (B) (F) 1-0 +F 2-0 +F 3-0 Linear relationship between F1 / (F2+F3) and UA concentration;

[0029] Figure 8 TCDs exhibit (A) selectivity and (B) resistance to interference of small molecules such as UA, anions and cations and various amino acids;

[0030] Figure 9 (A) Visual recognition of uric acid (UA) using TCDs with the assistance of a smartphone; (B) Manufacturing process and visual sensing performance of a paper-based uric acid sensor; (C) Linear relationship between the B / G ratio of the smartphone-integrated sensor and uric acid concentration; (D) Linear relationship between the B / G ratio of the test strip and uric acid concentration, and variation of uric acid concentration in the range of 0.0~500.0μM (all photos were taken under 365nm ultraviolet light). Detailed Implementation

[0031] The technical solution of the present invention will be further described below with reference to the embodiments.

[0032] Example 1

[0033] The triple-emission carbon quantum dot of the present invention is prepared by the following steps:

[0034] 0.052 g MPD (0.48 mol), 0.065 g (0.48 mol) malic acid, and 0.217 g Gd(NO3)3·6H2O (0.48 mol) were dissolved in 20.0 mL of a mixed solvent consisting of anhydrous ethanol and ultrapure water in a 1:1 volume ratio. The mixture was then sonicated for 10.0 min to ensure complete dissolution of all precursors. The resulting homogeneous solution was then transferred to a PTFE-lined stainless steel autoclave and heated at a constant temperature of 180°C for 12 h. After the reaction was complete, the autoclave was allowed to cool naturally to room temperature. The reaction product was then transferred to 50.0 mL centrifuge tubes and centrifuged at 10,000 rpm for 15.0 min to remove large particles and impurities. The supernatant containing TCDs was collected and further purified by continuous dialysis in 1.0 L ultrapure water (temperature: room temperature; dialysis medium: ultrapure water; molecular weight cutoff of dialysis bag: 100-500 D) for 3 days, with the ultrapure water being replaced every 24 h to ensure effective removal of small molecule impurities. Finally, the purified yellow TCDs solution was freeze-dried to obtain dried TCDs powder.

[0035] Example 2

[0036] Based on the example, the molar amount of Gd(NO3)3·6H2O was changed to 0.96 mmol (molar ratio of p-phenylenediamine (MPD): malic acid: gadolinium nitrate 1:1:2), and carbon quantum dots were obtained with other conditions unchanged.

[0037] Example 3

[0038] Based on the example, the molar amount of Gd(NO3)3·6H2O was changed to 1.44 mmol (the molar ratio of p-phenylenediamine (MPD): malic acid: gadolinium nitrate was 1:1:3), and carbon quantum dots were obtained with other conditions unchanged.

[0039] Example 4

[0040] Based on the example, the molar amount of Gd(NO3)3·6H2O was changed to 1.92 mmol (molar ratio of p-phenylenediamine (MPD): malic acid: gadolinium nitrate 1:1:4), and carbon quantum dots were obtained with other conditions unchanged.

[0041] Example 5

[0042] Based on the example, the molar amount of MPD was changed to 0.96 mmol (molar ratio of p-phenylenediamine (MPD): malic acid: gadolinium nitrate 2:1:1), and carbon quantum dots were obtained with other conditions unchanged.

[0043] Example 6

[0044] Based on the previous example, the molar amount of MPD was changed to 0.96 mmol, and the molar amount of Gd(NO3)3·6H2O was changed to 1.44 mmol (the molar ratio of p-phenylenediamine (MPD): malic acid: gadolinium nitrate was 2:1:3), while keeping other conditions unchanged, carbon quantum dots were obtained.

[0045] Example 7

[0046] Based on the example, the molar amount of MPD was changed to 1.44 mmol (molar ratio of p-phenylenediamine (MPD): malic acid: gadolinium nitrate 3:1:1), and carbon quantum dots were obtained with other conditions unchanged.

[0047] Example 8

[0048] Based on the previous example, the molar amount of MPD was changed to 1.44 mmol, and the molar amount of Gd(NO3)3·6H2O was changed to 0.96 mmol (the molar ratio of p-phenylenediamine (MPD): malic acid: gadolinium nitrate was 3:1:2), while keeping other conditions unchanged, carbon quantum dots were obtained.

[0049] Structural characterization

[0050] 1. TCDs element testing

[0051] The chemical composition of the TCDs prepared in Example 1 was evaluated by elemental analysis. As shown in Table 1, the TCDs contained hydrogen (H, 3.0%), carbon (C, 17.38%), nitrogen (N, 10.82%), and gadolinium (Gd) with a doping content of 50.0%. These results indicate that O, N, and Gd were successfully doped into the TCDs.

[0052] Table 1. Elemental composition of TCDs

[0053]

[0054] 2. Characterization of functional groups in TCDs

[0055] The functional groups of the TCDs prepared in Example 1 were characterized by Fourier transform infrared spectroscopy, and the results are as follows: Figure 1 As shown, in the range of 3000~3500 cm -1 The broad absorption bands appearing within this range are attributed to the stretching vibrations of OH and NH; 2610 cm⁻¹ -1 The absorption peak at 1620 cm⁻¹ is attributed to the stretching vibration of CH₄; -1 and 1370 cm -1 The peaks at 1075 cm⁻¹ correspond to C=O and C=N bonds, respectively; -1 and 670 cm -1 The absorption bands at these locations are associated with stretching vibrations of the CN bond and out-of-plane bending vibrations of the CH bond, respectively. Therefore, the hydrophilicity of TCDs is attributed to the unsaturated carbonaceous structure containing nitrogen and oxygen functional groups on their surface.

[0056] 3. Morphology and size testing of TCDs

[0057] The morphology and size of the TCDs prepared in Example 1 were measured using TEM, such as... Figure 2 As shown in Figure A, TCDs exhibit a uniform spherical morphology and excellent dispersibility in aqueous solution, with no obvious aggregation observed. Figure 2 B is the particle size distribution histogram of TCDs. The particle size distribution range of TCDs is 0.50~6.00 nm, and the average particle size is 2.63 ± 0.07 nm.

[0058] Performance testing

[0059] 1. Fluorescence performance test

[0060] The fluorescence properties of TCDs were studied by fluorescence spectroscopy, and the results are as follows: Figure 3 As shown.

[0061] like Figure 3 As shown in Figure A, the UV-Vis spectra of TCDs exhibit two typical characteristic peaks at 280 nm and 411 nm, which are attributed to the n-π* electronic transitions of nitrogen-containing functional groups on the surface of TCDs and the π-π* electronic transitions of the conjugated π system in the core, respectively.

[0062] Figure 3 B represents the emission spectra of TCDs at different excitation wavelengths. It can be seen that the emission spectra exhibit wavelength dependence; at an excitation wavelength of 300 nm, TCDs show relatively high fluorescence intensity at three distinct emission peaks at 346, 417, and 511 nm. The TCDs solution appears pale yellow under natural light. Figure 3 (Illustration B, left) appears bright cyan under 365 nm ultraviolet light. Figure 3 (Illustration of B on the right)

[0063] like Figure 3 As shown in Figure C, with quinine sulfate as a reference, the quantum yield of TCDs was measured to be 14.10%.

[0064] 2. Detection mechanism of UA by TCDs

[0065] The quenching effect of UA on the fluorescence of TCDs is a combination of internal filtration (IFE), dynamic quenching, and static quenching.

[0066] (1) Electrostatic adsorption mechanism

[0067] The 0.04 g mL sample prepared in Example 1 was tested. -1 TCDs and 0.04 g mL -1 The zeta potential of the TCDs mixed with 20.0 µM UA is shown in Table 2.

[0068] Table 2 Zeta potential of TCDs and TCDs / UA mixtures

[0069]

[0070] As shown in Table 2, the zeta potential of TCDs is 6.82 mV, while that of UA is -10.78 mV, and the zeta potential of the TCDs / UA mixture is -7.50 mV. No significant charge reversal was observed in this mixture, thus eliminating the possibility of electrostatic interactions.

[0071] (2) Dynamic / Quenching Mechanism

[0072] To investigate the fluorescence quenching mechanism of TCDs on UA, 2 mL of TCDs (0.04 mg / mL) were measured under 300 nm excitation. -1The fluorescence decay kinetics of a solution (pH=6) at the three characteristic emission peaks of 346, 417, and 511 nm were compared and analyzed before and after the addition of 20 μL UA. The results are shown in Table 3 and... Figure 4 As shown.

[0073] Table 3 Single-exponential fitting of TCDs and TCDs / UA decay curves

[0074]

[0075] Table 3 and Figure 4 As shown in Figure A, the blank TCDs system exhibits three characteristic emission peaks at 346, 417, and 511 nm, with corresponding fluorescence lifetimes of 3.72, 6.84, and 6.51 ns, respectively. Upon addition of 20.0 μM uric acid, the lifetime of the 346 nm peak significantly shortened to 2.61 ns, a sign of dynamic quenching. In contrast, the lifetimes of the 417 nm and 511 nm peaks showed only negligible changes (6.99 ns and 6.60 ns, respectively), indicating the presence of static quenching behavior.

[0076] (3) Internal filtration effect

[0077] Figure 4 As can be seen from B, the UV-Vis absorption spectrum of UA and the fluorescence excitation and emission spectra of TCDs have significant overlap, indicating that the fluorescence quenching mechanism between UA and TCDs is caused by the internal filtration effect.

[0078] 3. Luminescent performance test of TCDs prepared in Examples 1-8

[0079] The TCDs of this invention can produce three independent fluorescence emission peaks (at 346, 417, and 511 nm) at an excitation wavelength of 300 nm, according to (F 1-0 +F 2-0 +F 3-0 The fluorescence quenching efficiency is calculated as (F1 + F2 + F3) / (F3) as the scoring result. Where F... 1-0 F1 and F2 represent the fluorescence intensity of the emission peak at 346 nm before and after the addition of uric acid, respectively; F 2-0 F1 and F2 represent the fluorescence intensity of the emission peak at 417 nm before and after the addition of uric acid, respectively; F2 and F3 represent the fluorescence intensity of the emission peak at 417 nm before and after the addition of uric acid. 3-0 F1 and F2 represent the fluorescence intensity of the emission peak at 511 nm before and after the addition of uric acid, respectively.

[0080] Test method: Based on three-component TCDs synthesized from m-phenylenediamine, gadolinium nitrate hexahydrate, and malic acid in different molar ratios, a (F) assay was used for uric acid detection. 1-0 +F 2-0 +F3-0 The value of (F1+F2+F3) / (F3) (λex=300 nm). The concentration of TCDs was 0.04 mg·mL. -1 The system pH was 6.0, and the added uric acid concentration was 20.0 μM.

[0081] Test results are as follows Figure 5 As shown in Table 4.

[0082] Table 4 shows the scoring results for the fluorescence quenching efficiency of TCDs.

[0083]

[0084] Depend on Figure 5 It can be seen that TCDs prepared by different ratios of m-phenylenediamine, gadolinium hexahydrate and malic acid all exhibit their own unique luminescent properties.

[0085] From Table 4, it can be seen that, based on the quenching efficiency (F... 1-0 +F 2-0 +F 3-0 The ratio (F1 + F2 + F3) was used as a reference value, with the highest reference value observed for TCDs prepared by mixing the three precursors in equal proportions. Therefore, TCDs synthesized under optimized conditions with a molar ratio of 1:1:1 were selected as the fluorescent probes with the best luminescence performance.

[0086] 4. Effects of TCD concentration, pH, and reaction time on detection

[0087] The effect of TCDs concentration on the detection performance of UA was investigated. To explore the effect of TCDs concentration on the detection performance of UA, the fluorescence intensity of TCDs solutions (pH=6) prepared in Example 1 at different concentrations was measured at the three characteristic emission peaks of 346, 417, and 511 nm before and after the addition of 20 μL of UA, under an excitation wavelength of 300 nm. The fluorescence intensity was then measured using the quenching efficiency (F...). 1-0 +F 2-0 +F 3-0 The value of (F1+F2+F3) was used as a reference value and as an evaluation index for quantitative analysis.

[0088] pH Effect Test Method: To investigate the effect of TCDs on the detection performance of UA under different pH conditions, the TCDs solutions prepared in Example 1 (0.04 mg / mL) were measured at different pH conditions under an excitation wavelength of 300 nm. -1 The fluorescence intensity at the three characteristic emission peaks of 346, 417, and 511 nm was measured before and after adding 20 μL of UA; and the quenching efficiency (F) was used as the metric. 1-0 +F 2-0 +F 3-0The value of (F1+F2+F3) was used as a reference value and as an evaluation index for quantitative analysis.

[0089] Effect of reaction time on test method: To investigate the time required for TCDs to detect UA, the reaction time of the TCDs solution (0.04 mg / mL) prepared in Example 1 was measured at a 300 nm excitation wavelength. -1 The fluorescence intensity at the three characteristic emission peaks of 346, 417, and 511 nm (pH=6) before and after adding 20 μL UA was measured; and the quenching efficiency (F) was used as the quenching efficiency. 1-0 +F 2-0 +F 3-0 The value of (F1+F2+F3) was used as a reference value and as an evaluation index for quantitative analysis.

[0090] Test results are as follows Figure 6 As shown.

[0091] like Figure 6 As shown in Figure A, when the concentration of TCDs (prepared in Example 1) increased from 0.01 to 1.0 mg / mL... -1 When this occurs, the fluorescence quenching efficiency (F) is reduced. 1-0 +F 2-0 +F 3-0 The ratio (F0 / (F1+F2+F3)) (where F0 and F represent the fluorescence intensity of TCDs before and after mixing with UA) showed a significant change, with concentrations ranging from 0.01 to 0.04 mg / mL. -1 The quenching efficiency of TCDs gradually increases, especially at concentrations above 0.04 mg / mL. -1 The quenching efficiency gradually decreases. Its maximum value is 0.04 mg / mL. -1 Therefore, 0.04 mg / mL was chosen. -1 The optimal concentration of TCDs is [value missing].

[0092] like Figure 6 As shown in B, when the pH value (PBS solution, 10.0 mM) changes between 3.0 and 12.0, (F 1-0 +F 2-0 +F 3-0 The ratio (F1+F2+F3) showed significant changes, with the quenching efficiency of TCDs gradually increasing from pH 3 to 6 and gradually decreasing from pH 3 to 6. The highest value was achieved at pH 6.0. Therefore, pH 6.0 was selected as the optimal sensing condition for further research.

[0093] like Figure 6 C shows, (F) 1-0 +F 2-0 +F 3-0The value of 1 / (F1+F2+F3) increases sharply within 1.0 min and tends to be constant as time goes on, indicating that the sensing system has a fast response speed and can complete the response in only 1.0 min. Therefore, 1.0 min is adopted as the optimal response time in subsequent detection work.

[0094] 5. Validation of analytical methods

[0095] (1) Linear range

[0096] To investigate the effect of UA on the fluorescence intensity of TCDs (prepared in Example 1), a concentration of 0.04 mg / mL was prepared using 10 mM PBS buffer (pH 6.0). -1 A TCDs solution was prepared, and then different concentrations of UA were added to the prepared TCDs solution. After reacting for 1.0 min, the fluorescence intensity of the mixed solution at 346, 417, and 511 nm under 300 nm excitation was recorded. The results are as follows: Figure 7 As shown

[0097] like Figure 7 As shown in Figure A, in a TCDs solution (0.04 mg / mL) -1 When different concentrations of UA were added to TCDs (pH 6.0), the fluorescence intensity of the TCDs gradually decreased with increasing UA concentration. When the UA concentration reached 450.0 µM, the fluorescence of the TCDs was quenched by nearly 97.03%. This indicates that UA can effectively quench the fluorescence intensity of TCDs. Figure 7 As shown in B, the fluorescence quenching efficiency (F) 1-0 +F 2-0 +F 3-0 The equation y = 0.0196x + 0.9083 is linearly related to the concentration of UA, and the corresponding regression equation is y = 0.0196x + 0.9083. The detection limit is calculated to be 0.054 µM.

[0098] The detection limit of this method was compared with that of reported CDs-based fluorescence methods, as shown in Table 5. The sensitivity of this method is higher than that of any existing CDs-based fluorescence detection method.

[0099] Table 5 Detection performance of UA fluorescence method based on CDs

[0100]

[0101] (2) Selectivity and anti-interference performance

[0102] To test the selectivity of TCDs (prepared in Example 1) for UA, UA was mixed with different types of interfering substances, including metal cations (Mg). 2+ Na +Ba 2+ Fe 3+ Pb 2+ K + ), anion (I - F - C2O4 2- NO3 - SO4 2- HSO3 - ,Br - HPO4 2- H2PO4 - Cl - The following amino acids (DL-cysteine, phenylalanine, serine, tyrosine, glycine) and other potentially interfering compounds (glutamate, urea, ascorbic acid, sucrose, glucose, fructose, adenine, creatinine, allopurinol, hypoxanthine) were prepared as 10.0 mM solutions. 20.0 μL of each solution was added to a solution containing 2.0 mL of TCDs (0.04 mg / mL). -1 The solution was mixed thoroughly in a 5.0 mL cuvette (pH 6.0). The maximum fluorescence intensity of the mixed solution was recorded at 346, 417, and 511 nm at an excitation wavelength of 300 nm.

[0103] To test the anti-interference ability of TCDs (prepared in Example 1) for UA detection, a series of 20.0 μL UA solutions (10.0 mM) were added to solutions containing 2.0 mL TCDs (0.04 mg / mL). -1 The mixture was placed in a 5.0 mL cuvette (pH 6.0). Then, 2.0 μL of the aforementioned interfering substance was added to each cuvette to bring the final concentration to 0.1 mM. Similar to the selectivity study, the fluorescence intensity of the mixture solution at 346, 417, and 511 nm was recorded under 300 nm excitation.

[0104] Test results are as follows Figure 8 As shown.

[0105] like Figure 8 As shown in Figure A, the introduction of UA leads to a sharp decrease in the fluorescence intensity of TCDs, while other potentially interfering substances have almost no significant effect on the fluorescence intensity of TCDs, indicating that the detection method proposed in this work has excellent selectivity for UA detection.

[0106] like Figure 8 As shown in Figure B, the fluorescence of the TCDs / UA system is not affected by the aforementioned foreign substances, indicating that the method has good anti-interference ability. Therefore, the proposed method has the potential to be used for the determination of UA in actual urine samples.

[0107] (3) Feasibility verification of testing

[0108] Smartphone-assisted UA visual sensing: Validating the feasibility of a three-RFR platform based on TCDs (prepared in Example 1) combined with smartphone colorimetry for uric acid detection.

[0109] Under 365 nm ultraviolet light irradiation, when the uric acid concentration varied within the range of 0.0–500.0 μM, the TCDs (prepared in Example 1) solution (0.04 mg / mL) showed different results. -1 (pH=6) exhibits a distinct color gradient from bright cyan to royal blue, and the color intensity is closely related to the uric acid concentration, such as... Figure 9 As shown in (A). The image signal was converted into RGB values ​​using the ColorColl App on a smartphone, and the blue / green channel ratio (B / G) was used as a quantitative indicator. The results showed that the B / G ratio had a good linear relationship with uric acid concentration, with the linear equation being B / G = 0.0048[UA] + 1.0623, and the correlation coefficient R. 2 =0.9941, detection limit is 2.20 μM, such as Figure 9 As shown in (C), this portable smartphone integrates an RFR system that enables the visualization and quantitative detection of uric acid.

[0110] Uric acid test strips based on TCDs: The analytical performance of the prepared fluorescent paper sensor was evaluated by testing solutions of TCDs (prepared in Example 1) containing different concentrations of uric acid (UA) at 0.04 mg / mL. -1 A uric acid solution (pH=6) was added to a test strip (qualitative filter paper). Under 365 nm UV light, as the uric acid concentration increased from 0.0 to 500.0 μM, the fluorescence of the TCDs-modified test strip gradually changed from bright cyan to royal blue. Figure 9 (B) shows the color change, which is consistent with that in aqueous solution, indicating that the optical properties of TCDs are well preserved after being fixed on the paper base, and the fluorescence quenching phenomenon is obvious, with more significant fluorescence attenuation at higher uric acid concentrations. Quantitative analysis of the RGB values ​​of the test strip collected by a smartphone APP showed that the G / B ratio and uric acid concentration were linearly related in the range of 0.0~500.0 μM, with a linear regression equation of G / B = 0.0019[UA] +1.0892 and a detection limit of 5.61 μM. This confirms that fluorescent paper loaded with TCDs can be used for the quantitative detection of uric acid. Figure 9 As shown in (D).

[0111] (4) Actual sample testing

[0112] The actual samples were drug-free urine samples collected from four healthy human volunteers. Each urine sample was centrifuged at 10,000 rpm / min for 10.0 min at room temperature to remove suspended particles, cell debris, and proteins that might interfere with the fluorescence reaction. After centrifugation, the clear supernatant was carefully collected and further diluted 10-fold with ultrapure water. The TCDs solution (0.04 mg / mL) was then added. -1 The sample extract (pH 6.0) was transferred to 5.0 mL cuvettes, each containing 2.0 mL of TCDs solution. 20.0 μL of sample extract was added and mixed. The fluorescence intensity (F0 and F) of the TCDs solution before and after adding the sample extract was measured at 346, 417, and 511 nm at an excitation wavelength of 300 nm.

[0113] Meanwhile, in the experiment for determining UA in urine samples based on high performance liquid chromatography (HPLC), a Nova-Pak C18 column (150 mm × 3.9 mm inner diameter; Waters Associates) was used. The mobile phase consisted of 0.2% acetic acid-methanol (94:6 v / v) at a flow rate of 1.0 mL / min. -1 Column temperature: 30 °C, detection wavelength: 288 nm, injection volume: 10 μL. The buffer solution was filtered under reduced pressure using a 0.45 μm HATF 04700 membrane (Millipore). The filtered mobile phase was then degassed in an ultrasonic bath to remove dissolved air and stored for later use. The assay was repeated three times, and the average value was taken to ensure accuracy. The test results are shown in Table 6.

[0114] Table 6. Detection of UA in urine samples

[0115]

[0116] As shown in Table 6, the UA concentrations in the urine samples were 320.14, 277.21, 307.76, and 347.82 μM, respectively. Spiking recovery experiments were conducted by adding uric acid standard solutions at three concentration levels (30.0, 60.0, and 90.0 μM) to real urine samples. The average recovery rate ranged from 95.4% to 103.4%, with relative standard deviations (RSDs) ≤2.29%. Furthermore, HPLC analysis showed consistency between the detection results of the two methods, indicating that the proposed detection method has high accuracy.

Claims

1. A method for preparing triple-emission carbon quantum dots, characterized in that, Includes the following steps: (1) Add p-phenylenediamine, malic acid and gadolinium nitrate to a solvent to dissolve all raw materials to obtain a precursor solution; (2) A hydrothermal reaction is carried out. After the reaction is completed, large particulate impurities and small molecule impurities are removed, and the carbon quantum dot powder is dried to obtain the powder. The molar ratio of p-phenylenediamine, malic acid and gadolinium nitrate is 1~3:1:1~4.

2. The method for preparing triple-emission carbon quantum dots according to claim 1, characterized in that, The molar ratio of p-phenylenediamine, malic acid, and gadolinium nitrate is 1~2:1:

1.

3. The method for preparing triple-emission carbon quantum dots according to claim 1, characterized in that, The hydrothermal reaction was carried out at a temperature of 180°C for 12 hours.

4. The method for preparing triple-emission carbon quantum dots according to claim 1, characterized in that, The solvent is a mixture of ethanol and water.

5. A triple-emission carbon quantum dot prepared by any one of claims 1 to 4.

6. The triple-emission carbon quantum dot according to claim 5, characterized in that, The triple-emission carbon quantum dot has three distinct emission peaks at 346, 417, and 511 nm.

7. The triple-emission carbon quantum dot according to claim 5, characterized in that, The excitation wavelength of the triple-emission carbon quantum dot is 300 nm.

8. A test paper comprising the triple-emission carbon quantum dots as described in any one of claims 5 to 7.

9. The application of the triple-emission carbon quantum dot of claim 5 or the test strip of claim 8 in the rapid detection of uric acid.

10. The application according to claim 9, characterized in that, The detection limit for uric acid is 0.054 µM.

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