Copper-doped carbon dots with catechol oxidase-like activity as well as preparation method and application of copper-doped carbon dots
By using copper-doped carbon dot nanozymes to catalyze the oxidation of catecholamines, the problems of expensive equipment, complex operation, and poor stability of natural enzymes in the detection of catecholamines in existing technologies have been solved, and highly sensitive, rapid, and visual colorimetric detection has been achieved.
Patent Information
- Application Number
- CN202511652978.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-12
- Publication Date
- 2026-01-27
AI Technical Summary
Existing methods for detecting catecholamines suffer from problems such as expensive equipment, complex operation, long processing time, low sensitivity, poor stability of natural enzymes, and high cost, which limit their application in routine testing and on-site immediate use.
Copper-doped carbon dots were used as nanozymes to detect catecholamines by colorimetric method. The catechol oxidase-like activity of copper-doped carbon dots was utilized to achieve highly sensitive, rapid and visualized detection.
Highly sensitive detection of catecholamines was achieved with a detection limit of 0.489 μM. The solution color changed significantly during the detection process, and the repeatability was good. Furthermore, the copper-doped carbon dots showed low cytotoxicity and good biocompatibility.
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Figure CN121402144A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of catecholamine detection technology, specifically to a copper-doped carbon dot with catechol oxidase-like activity, its preparation method, and its application. Background Technology
[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.
[0003] Catecholamines are a class of neurotransmitters containing catechols and amino groups, including adrenaline, noradrenaline, dopamine, and their derivatives; these substances are all important and typical adrenergic receptor agonists. Catecholamines regulate basic physiological functions and transmit physiological signals in the body, acting as important signaling mediators in normal physiological processes. Their levels also change accordingly during pathological processes. Clinically, they can be used to assist in the diagnosis of endocrine-related diseases such as hypertension, hyperthyroidism, pheochromocytoma, and neuroblastoma. Therefore, establishing simple, sensitive, and accurate methods for analyzing and detecting catecholamines is essential.
[0004] Currently, methods for detecting catecholamines include high-performance liquid chromatography-electrochemical methods and ion chromatography. These methods generally require large instruments and are relatively complex to operate. They are expensive, time-consuming, and have low sensitivity, making it difficult to meet daily detection requirements. This greatly limits the promotion and application of these detection methods and increases the cost for users.
[0005] Enzyme-linked colorimetric assays based on natural enzymes are widely used due to their high specificity and good sensitivity. However, natural enzymes have drawbacks such as high preparation costs, poor stability, demanding storage conditions, and easy inactivation, which limit their large-scale and on-site real-time detection applications.
[0006] To overcome the bottlenecks in the practical detection applications of natural enzymes, nanozymes, as a class of functional nanomaterials with enzyme-like catalytic activity, have been widely introduced into the fields of biosensing and in vitro diagnostics due to their high stability, low cost, and scalable preparation. Summary of the Invention
[0007] To overcome the above problems, this invention provides copper-doped carbon dots with catechol oxidase-like activity, their preparation method, and applications. The copper-doped carbon dots provided by this invention can act as nanozymes to specifically catalyze the oxidation of catecholamines, enabling the colorimetric detection of catecholamines.
[0008] To achieve the above technical objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a method for preparing copper-doped carbon dots, comprising the following steps: Disodium guanylate and divalent copper salt were dispersed in water, heat-treated, and the liquid was collected by centrifugation. After dialyzing and drying, copper-doped carbon dots were obtained.
[0009] In one or more embodiments, the divalent copper salt includes one of copper chloride, copper sulfate, copper nitrate, and copper acetate, preferably copper chloride.
[0010] In one or more embodiments, the molar ratio of disodium guanylate to divalent copper salt is (1~2):1.
[0011] In one or more embodiments, the concentration of the divalent copper salt in water is 0.2~0.8 mol / L.
[0012] In one or more embodiments, the heat treatment temperature is 140~200 °C, and the heat treatment time is 6~12 h.
[0013] In one or more embodiments, the pH of the solution is adjusted to 9-11 before heat treatment.
[0014] In one or more embodiments, the molecular weight cutoff for dialysis is 500-1500 Da, preferably 500-1000 Da; the dialysis time is 24-48 h.
[0015] In a second aspect, the present invention provides a copper-doped carbon dot, which is prepared by the preparation method described in the first aspect.
[0016] A third aspect of the present invention provides the application of the copper-doped carbon dots described in the second aspect in the detection of catecholamine content.
[0017] A fourth aspect of the present invention provides the application of the copper-doped carbon dots described in the second aspect in the preparation of products for detecting catecholamine content.
[0018] A fifth aspect of the present invention provides a method for detecting catecholamine content, comprising the following steps: Copper-doped carbon dots were mixed with the sample to be tested, and after incubation in the dark, a mixture was obtained. The absorbance of the mixture at 485 nm was measured. The catecholamine content in the sample was obtained based on a standard curve of catecholamine concentration versus absorbance value.
[0019] In one or more embodiments, the incubation time is 10 to 20 minutes, preferably 15 minutes.
[0020] The beneficial effects of this invention are as follows: (1) The copper-doped carbon dots provided by the present invention can be used as nanozymes to specifically catalyze the oxidation of catecholamines, enabling the detection of catecholamines by colorimetric method. The copper atoms in the copper-doped carbon dots are dispersed in the form of single atoms, and the local coordination structure is Cu-N3O, which is similar to the metal active sites in natural enzymes. This structure significantly enhances the electron transfer ability and redox activity of the Cu center, making it exhibit high catalytic activity and substrate specificity in the oxidation reaction of catecholamines.
[0021] (2) The copper-doped carbon dots prepared by using disodium guanylate as a carbon source and copper chloride as a metal source in this invention overcome the difficulties of complex design ideas and cumbersome preparation process of traditional multifunctional nanomaterials. Moreover, copper-doped carbon dots have abundant surface active groups and excellent biocompatibility, with little toxic side effects on normal cells and tissues.
[0022] (3) The copper-doped carbon dots provided by this invention can achieve highly sensitive, rapid, and visual colorimetric detection of adrenaline, with a detection limit of 0.489 μM. In the range of 0-100 μg / mL, the absorbance shows a good linear relationship with the adrenaline concentration. During the detection process, the solution gradually changes from colorless to brownish-yellow, with obvious response and good repeatability.
[0023] (4) The size of the copper-doped carbon dots prepared by the present invention is about 1.8 nm, and their size and catalytic activity can be adjusted by controlling the ratio of guanylic acid to divalent copper salt, solution pH, hydrothermal reaction time, reaction temperature, etc. Attached Figure Description
[0024] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.
[0025] Figure 1 This is a transmission electron microscope (TEM) image of the copper-doped carbon dots prepared in Example 4; Figure 2 The image is a high-angle annular dark-field scanning transmission electron microscope (HAADF-STEM) image of the copper-doped carbon dots prepared in Example 4. Figure 3 The results of synchrotron radiation for the copper-doped carbon dots prepared in Example 4; Figure 4 The UV-Vis absorption spectrum of the oxidation reaction of 3,5-di-tert-butylcatechol catalyzed by copper-doped carbon dots; Figure 5 The double reciprocal kinetic curves for the oxidation of 3,5-di-tert-butylcatechol catalyzed by copper-doped carbon dots; Figure 6 This is a standard curve diagram of the copper-doped carbon dot cores prepared in Example 4 of the present invention for the colorimetric detection of adrenaline; Figure 7 The results show the cytotoxicity of copper-doped carbon dots on mouse fibroblast L929 cells. Detailed Implementation
[0026] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.
[0027] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.
[0028] Enzyme-linked colorimetric assays based on natural enzymes are widely used due to their high specificity and good sensitivity. However, natural enzymes suffer from drawbacks such as high preparation costs, poor stability, demanding storage conditions, and easy inactivation, limiting their large-scale and on-site point-of-care detection applications. To overcome the bottlenecks in practical detection applications of natural enzymes, nanozymes, as a class of functional nanomaterials with enzyme-like catalytic activity, have been widely introduced into the fields of biosensing and in vitro diagnostics due to their high stability, low cost, and scalable preparation capabilities.
[0029] This invention first tests the catalytic performance of copper-doped carbon dots as nanoenzymes by catalyzing the oxidation of 3,5-di-tert-butylcatechol. The final product, 3,5-di-tert-butylcatechone, is consistent with the natural enzyme system, confirming that the copper-doped carbon dots possess catechol oxidase-like activity. Simultaneously, kinetic analysis results show that the Kx of the reaction… m It is 0.22 mM, V m 1.001 mM·s -1 The catalytic process requires the participation of O2, which proves that copper-doped carbon dots have good catechol oxidase activity and stable catalytic kinetics.
[0030] The copper-doped carbon dots provided by this invention can serve as nanozymes for the specific catalytic oxidation of catecholamines, enabling colorimetric detection of catecholamines. In the copper-doped carbon dots, copper atoms are dispersed in single-atom form, with a local coordination structure of Cu-N3O, similar to the metal active sites in natural enzymes. This structure significantly enhances the electron transfer capacity and redox activity of the Cu centers, resulting in highly efficient catalytic activity and substrate specificity in the catecholamine oxidation reaction.
[0031] The copper-doped carbon dots provided by this invention enable highly sensitive, rapid, and visual colorimetric detection of adrenaline, with a detection limit of 0.489 μM. Within the range of 0-100 μg / mL, the absorbance exhibits a good linear relationship with adrenaline concentration. During detection, the solution gradually changes from colorless to brownish-yellow, demonstrating a clear response and good repeatability.
[0032] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.
[0033] Example 1 Preparation of copper-doped carbon dots: Dissolve 3.84 mmol of disodium guanylate in 15 mL of pure water, then add 3.84 mmol of copper chloride pentahydrate and stir for 30 min until completely dissolved. Adjust the pH of the mixture to 9.5. Transfer the mixture to a reaction vessel and heat-treat at 160 °C for 6 h. After the reaction vessel temperature has completely cooled to room temperature, centrifuge at 10,000 rpm for 10 min to collect the supernatant. Repeat the centrifugation operation 3 times. Subsequently, dialyze the crude copper-doped carbon dots for 24 h using a dialysis bag with a molecular weight cutoff of 1000 Da to remove impurity ions from the solution. After freeze-drying, purified copper-doped carbon dots are obtained.
[0034] Example 2 Preparation of copper-doped carbon dots: Dissolve 5 mmol of disodium guanylate in 10 mL of pure water, then add 2.5 mmol of copper chloride pentahydrate and stir for 30 min until completely dissolved. Adjust the pH of the mixture to 11. Transfer the mixture to a reaction vessel and heat-treat at 200 °C for 10 h. After the reaction vessel temperature has completely cooled to room temperature, centrifuge at 10000 rpm for 10 min to collect the supernatant. Repeat the centrifugation operation 3 times. Subsequently, dialyze the crude copper-doped carbon dots for 24 h using a dialysis bag with a molecular weight cutoff of 1000 Da to remove impurity ions from the solution. After freeze-drying, purified copper-doped carbon dots are obtained.
[0035] Example 3 Preparation of copper-doped carbon dots: 7.69 mmol of disodium guanylate was dissolved in 13 mL of pure water, and then 3.84 mmol of copper chloride pentahydrate was added. The mixture was stirred for 30 min until completely dissolved, and the pH of the mixture was adjusted to 10. The mixture was transferred to a reaction vessel and heat-treated at 180 °C for 12 h. After the reaction vessel temperature had completely cooled to room temperature, the supernatant was collected by centrifugation at 10,000 rpm for 10 min, and the centrifugation was repeated 3 times. Subsequently, the crude copper-doped carbon dots were dialyzed for 24 h using a dialysis bag with a molecular weight cutoff of 1000 Da to remove impurity ions from the solution. The purified copper-doped carbon dots were obtained after freeze-drying.
[0036] Example 4 Preparation of copper-doped carbon dots: Dissolve 7.69 mmol of disodium guanylate in 13 mL of pure water, then add 7.69 mmol of copper chloride pentahydrate and stir for 30 min until completely dissolved. Adjust the pH of the mixture to 10.23. Transfer the mixture to a reaction vessel and heat-treat at 180 °C for 8 h. After the reaction vessel temperature has completely cooled to room temperature, centrifuge at 10,000 rpm for 10 min to collect the supernatant. Repeat the centrifugation operation 3 times. Subsequently, dialyze the crude copper-doped carbon dots for 24 h using a dialysis bag with a molecular weight cutoff of 1000 Da to remove impurity ions from the solution. After freeze-drying, purified copper-doped carbon dots are obtained.
[0037] Figure 1 The image shown is a transmission electron microscope (TEM) image of copper-doped carbon dots obtained in this embodiment. Figure 1 It can be seen that the size of the copper-doped carbon dots is about 1.8 nm and they are uniformly dispersed.
[0038] Figure 2 The image obtained in this embodiment is a high-angle annular dark-field scanning transmission electron microscope (HAADF-STEM) image of copper-doped carbon dots. Figure 2 As can be seen from the figure, the copper-doped carbon dots appear in the form of single copper atoms, and the ones highlighted in yellow are copper atom pairs.
[0039] Figure 3 The synchrotron radiation results of copper-doped carbon dots obtained in this embodiment are from... Figure 3 As can be seen, Cu exists in a Cu-N3O coordination structure without Cu-Cu bonds. Combining aberration-corrected electron microscopy and DFT simulation results, the coordination structure of the copper-doped carbon dots is determined to be N3-Cu-O-Cu-N3. In the copper-doped carbon dots, copper atoms are dispersed in single-atom form, with a local coordination structure of Cu-N3O, similar to the metal active sites in natural enzymes. This structure significantly enhances the electron transfer ability and redox activity of the Cu center.
[0040] Example 5 Using the copper-doped carbon dots prepared in Example 4 as the test object, their enzyme-like catalytic performance for the oxidation reaction of 3,5-di-tert-butylcatechol was tested.
[0041] 3,5-Di-tert-butylcatechol and copper-doped carbon dots were mixed in 1 mL of PBS buffer. The final concentrations of 3,5-di-tert-butylcatechol and copper-doped carbon dots were 0.5 mM and 0.5 mg / mL, respectively. The reaction was carried out at room temperature for 8 min. The absorbance of the final reaction solution was measured, and the results are as follows: Figure 4 As shown, the reaction system exhibits a significant absorption peak at approximately 415 nm, and the absorption intensity gradually increases with increasing reaction time, indicating the continuous formation of the product 3,5-di-tert-butylbenzoquinone. This result demonstrates that copper-doped carbon dots can effectively catalyze the oxidation of 3,5-di-tert-butylcatechol, exhibiting stable and sustainable catechol oxidase activity.
[0042] like Figure 5 As shown, the copper-doped carbon dot-catalyzed reaction of 3,5-di-tert-butylcatechol conforms to a typical Michaelis-Menten kinetic model. The Michaelis constant K for the catalytic reaction is calculated from the slope and intercept. m The maximum reaction rate V is 0.22 mM. m 1.001 mM·s -1 The results confirmed that the copper-doped carbon dot material prepared in this invention has good catechol oxidase activity and stable catalytic kinetics.
[0043] Example 6 Using the copper-doped carbon dots prepared in Example 4 as the test object, the material was used for colorimetric detection of adrenaline.
[0044] Epinephrine was mixed with copper-doped carbon dots in 1 mL of PBS buffer, resulting in final concentrations of 0.5 mg / mL for the copper-doped carbon dots and 0-100 µg / mL for the epinephrine. -1 After mixing thoroughly, the mixture was incubated at 37 °C in the dark for 15 min, and then the absorbance change was recorded at 480 nm. The results are as follows. Figure 6 As shown, absorbance exhibits a good linear relationship with adrenaline concentration within the range of 0-100 μg / mL. The inset shows the solution color gradually changing from colorless to brownish-yellow, corresponding to the formation of the product adrenaline. The detection limit is 0.489 μM, indicating that copper-doped carbon dots can achieve highly sensitive, rapid, and visual colorimetric detection of adrenaline.
[0045] Example 7 Cytotoxicity assay: Cell viability / deadness staining was performed using a commercial Calcein-AM / PI kit (operated according to the instructions). Green indicates live cell signal and red indicates dead cell signal.
[0046] Using the copper-doped carbon dots prepared in Example 4 as the test object, by Figure 7 As can be seen, with the increase of material concentration (0.25~0.75 mg / mL), the overall morphology of L929 cells remained intact, and the green fluorescence signal was significantly stronger than the red fluorescence signal, indicating that most cells were still in an active state, with only a small amount of red signal appearing at the high concentration of 0.75 mg / mL. The results show that the copper-doped carbon dots prepared in this invention have low toxicity to L929 cells and good biocompatibility.
[0047] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.
Claims
1. A method for preparing copper-doped carbon dots, characterized in that, Includes the following steps: Disodium guanylate and divalent copper salt were dispersed in water, heat-treated, and the liquid was collected by centrifugation. After dialyzing and drying, copper-doped carbon dots were obtained.
2. The preparation method according to claim 1, characterized in that, The divalent copper salt includes one of copper chloride, copper sulfate, copper nitrate, and copper acetate, preferably copper chloride; Alternatively, the molar ratio of disodium guanylate to divalent copper salt is (1~2):
1.
3. The preparation method according to claim 1, characterized in that, The concentration of divalent copper salts in water is 0.2~0.8 mol / L.
4. The preparation method according to claim 1, characterized in that, The heat treatment temperature is 140~200 ℃, and the heat treatment time is 6~12 h.
5. The preparation method according to claim 1, characterized in that, Before heat treatment, adjust the pH of the solution to 9-11.
6. The preparation method according to claim 1, characterized in that, The molecular weight cutoff for dialysis is 500-1500 Da, preferably 500-1000 Da; the dialysis time is 24-48 h.
7. A copper-doped carbon dot, characterized in that, It is prepared by the preparation method according to any one of claims 1 to 6.
8. The application of the copper-doped carbon dots as described in claim 7 in the detection of catecholamine content.
9. The application of the copper-doped carbon dots as described in claim 7 in the preparation of products for detecting catecholamine content.
10. A method for detecting catecholamine content, characterized in that, Includes the following steps: Copper-doped carbon dots were mixed with the sample to be tested, and after incubation in the dark, a mixture was obtained. The absorbance of the mixture at 485 nm was measured. The catecholamine content in the sample was obtained based on a standard curve of catecholamine concentration versus absorbance value.