Copper-crosslinked carbon dot hydrogel nanozymes, their preparation methods, and their application in the detection of tert-butylhydroquinone.

Abstract

This invention discloses a copper-crosslinked carbon dot hydrogel nanozyme, its preparation method, and its application in the detection of tert-butylhydroquinone, belonging to the field of optical sensing technology. The preparation method uses 1,2,3,4-butanetetracarboxylic acid and citric acid as raw materials, and prepares carbon dots in one step via a hydrothermal method, followed by the application of Cu... 2+ A copper-crosslinked carbon dot hydrogel nanozyme was obtained using this invention as the catalytic center. The copper-crosslinked carbon dot hydrogel nanozyme prepared in this invention exhibits high peroxidase-mimicking activity, oxidizing the peroxidase substrate dopamine to reddish-brown aminochrome in the presence of H₂O₂, thus serving as a novel peroxidase-mimicking nanozyme. This invention also discloses a colorimetric sensor and a visualization sensor constructed based on the above-mentioned copper-crosslinked carbon dot hydrogel nanozyme for the detection of tert-butylhydroquinone.

Description

Technical Field

[0001] This invention belongs to the field of optical sensing technology, specifically relating to copper cross-linked carbon dot hydrogel nanozymes, their preparation methods, and their application in the detection of tert-butylhydroquinone. Background Technology

[0002] tert-butylhydroquinone (TBHQ) is a common phenolic food antioxidant that prevents food from oxidizing and spoiling. Due to its low toxicity and high thermal stability, it is widely used in the preservation of edible vegetable oils, fried foods, and nuts. However, the product TBBQ, formed after the oxidation of TBHQ, poses potential health risks, such as inducing apoptosis and cancer. Therefore, developing a simple, rapid, accurate, and sensitive method for detecting TBHQ in food is of great significance. Currently, common methods for detecting TBHQ include high-performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), capillary electrophoresis, and electrochemical methods. While these methods are accurate, sensitive, and have a certain specificity for TBHQ detection, they often have several drawbacks, such as high cost, long processing time, bulky and inconvenient instruments, and the need for skilled personnel. Therefore, developing an economical, simple, and rapid method for detecting tert-butylhydroquinone is essential.

[0003] Hydrogels are porous polymer gels with a three-dimensional network structure, typically containing polar functional groups such as carboxyl and hydroxyl groups. The three-dimensional porous network structure and abundant polar functional groups of hydrogels endow them with advantages such as large specific surface area, high porosity, good stability, abundant active sites, and good hydrophilicity. These advantages make hydrogels a promising material for various fields, such as analytical sensing and biomedicine. However, most commonly used hydrogels require chemical cross-linking agents during synthesis, and these cross-linking agents are often toxic. Furthermore, current research on hydrogels mainly focuses on noble metal hydrogels, such as gold, palladium, and silver hydrogels, which leads to high costs for large-scale synthesis. Therefore, the research and development of non-noble metal nanozyme-functionalized hydrogels that are easy to mass-produce, environmentally friendly, non-toxic, and low-cost is of great significance. Summary of the Invention

[0004] To address the shortcomings and challenges of existing technologies, this invention provides a copper-crosslinked carbon dot hydrogel nanozyme, its preparation method, and its application in the detection of tert-butylhydroquinone. It has the advantages of convenient detection, high sensitivity, speed, and good selectivity.

[0005] This invention is achieved through the following technical solution:

[0006] This invention provides a method for preparing copper-crosslinked carbon dot hydrogel nanozymes. The method uses 1,2,3,4-butanetetracarboxylic acid and citric acid as raw materials to prepare carbon dots in one step via a hydrothermal method, followed by the application of Cu... 2+ To obtain copper-crosslinked carbon dot hydrogel nanozymes (BC-CDs@Cu) as the catalytic center, the method includes the following steps:

[0007] S1. Add 315.2 mg of citric acid, 70.2-702 mg of 1,2,3,4-butanetetracarboxylic acid, and 20 mL of ultrapure water to the container in sequence, and sonicate for a period of time to mix evenly. Transfer to a 50 mL high-pressure reactor.

[0008] S2 and the mixture solution from step S1 are heated at 80–220 °C for 4–24 h. After cooling to room temperature, the mixture is centrifuged at 12000 rpm / min for 10 min, the supernatant is collected, filtered through a 0.22 μm filter membrane, the filtrate is freeze-dried, and then washed with solvent. The washing liquid is then evaporated using a rotary evaporator to remove the solvent and obtain dried carbon dots.

[0009] S3. Add an appropriate amount of water to the carbon dots obtained in step S2 to obtain a 2 mg / mL carbon dot aqueous solution, and adjust the pH of the solution with 5 M sodium hydroxide.

[0010] S4. Take 1000 volume parts of the solution from step S3, add 50-800 volume parts of 1M copper nitrate, shake well to obtain a sky blue hydrogel, centrifuge the hydrogel at 12000 rpm / min for 10 min, discard the supernatant, and wash three times with ultrapure water to obtain pure BC-CDs@Cu.

[0011] Preferably, in step S1, the amount of 1,2,3,4-butanetetracarboxylic acid is 281 mg; the ultrasonic time is 5 min; in step S2, the reaction temperature is 200℃; the heating time is 12 h; the solvent is acetonitrile; in step S3, the solution pH is 7; and in step S4, the amount of copper nitrate is 100 parts by volume.

[0012] The present invention also provides a copper crosslinked carbon dot hydrogel nanozyme prepared by the above method.

[0013] This invention also provides applications of the above-mentioned copper-crosslinked carbon dot hydrogel nanozyme, namely, copper-constructed colorimetric and visualization sensors for TBHQ detection.

[0014] The specific application method is as follows:

[0015] Colorimetric sensor construction:

[0016] (1) Preparation of multiple detection solutions: 100 volumes of phosphate buffer solution, 200 volumes of BC-CDs@Cu with a concentration of 4 mg / mL, 10 volumes of TBHQ solution with different concentrations, 100 volumes of dopamine with a concentration of 20 mM, and 60 volumes of H2O2 with a concentration of 100 mM were added and diluted with water to a total volume of 2 mL to obtain multiple detection solutions;

[0017] (2) Mix the test solutions of each group evenly and react at room temperature for 10 min;

[0018] (3) Measure the ultraviolet-visible absorption spectrum of the mixed solution prepared in step (2);

[0019] (4) TBHQ was detected based on the relationship between the absorption peak intensities of the dopamine oxidation products (aminochrome) corresponding to different concentrations of TBHQ at 480 nm.

[0020] As the concentration of TBHQ increases, the absorption peak intensity of aminochrome at 480 nm gradually decreases. The TBHQ concentration and the absorbance of aminochrome show a linear relationship in the range of 0.5-20 μM, with a detection limit of 290 nm.

[0021] b. Construction of Visual Detection Method:

[0022] (5) Preparation of multiple sets of test samples: 10 volumes of phosphate buffer solution, 160 volumes of BC-CDs@Cu, 20 volumes of TBHQ solution of different concentrations, 5 volumes of dopamine at a concentration of 40mM, and 6 volumes of H2O2 at a concentration of 100mM were added to 96 microplates to obtain multiple sets of test samples.

[0023] (6) Mix the test samples thoroughly and react at room temperature for 10 min;

[0024] (7) Take a picture of the sample obtained in step (6) using a smartphone to record the color change of each well plate;

[0025] (8) The RGB values ​​of the image obtained in step (7) are analyzed by the image processing software ImageJ to obtain the linear equation between TBHQ concentration and RGB value, thereby achieving the purpose of colorimetric detection of TBHQ.

[0026] As the concentration of TBHQ increases, the color of CDs-Cu hydrogel exhibits a gradient change. The B / R value increases with the increase of tert-butylhydroquinone concentration. The TBHQ concentration and the B / R value show a linear relationship in the range of 5-100 μM, and the detection limit is 1.1 μM.

[0027] Preferably, the phosphate buffer solution has a concentration of 0.2M and a pH of 7.

[0028] Compared with the prior art, the beneficial effects of the present invention include:

[0029] (1) This method uses 1,2,3,4-butanetetracarboxylic acid and citric acid as raw materials to prepare carbon dots in one step via a hydrothermal method, and then uses Cu 2+ A copper-crosslinked carbon dot hydrogel nanozyme was obtained using this as the catalytic center. This copper-crosslinked carbon dot hydrogel nanozyme exhibits high peroxidase-like activity and good stability.

[0030] (2) The copper cross-linked carbon dot hydrogel nanozymes prepared in this invention are used to construct colorimetric and visualization sensors for the detection of TBHQ, which have the advantages of being convenient, sensitive, fast and selective. Attached Figure Description

[0031] Figure 1 This is a photograph of the copper cross-linked carbon dot hydrogel nanozyme of the present invention;

[0032] Figure 2 This is a spectrum depicting the enzyme activity of the copper-crosslinked carbon dot hydrogel nanoenzyme of this invention.

[0033] Figure 3 UV-Vis absorption spectrum of tert-butylhydroquinone detected by copper-crosslinked carbon dot hydrogel nanozyme;

[0034] Figure 4 Visual detection of TBHQ based on copper-crosslinked carbon dot hydrogel nanozymes. Detailed Implementation

[0035] The present invention will be further described below with reference to the accompanying drawings.

[0036] Example 1: Preparation of copper cross-linked carbon dot hydrogel nanozymes

[0037] This method uses 1,2,3,4-butanetetracarboxylic acid and citric acid as raw materials to prepare carbon dots in one step via a hydrothermal method, followed by the addition of Cu. 2+ A copper-crosslinked carbon dot hydrogel nanozyme was obtained using it as the catalytic center.

[0038] S1. Add 315.2 mg of citric acid, 70.2-702 mg of 1,2,3,4-butanetetracarboxylic acid, and 20 mL of ultrapure water to the container in sequence, and sonicate for a period of time to mix evenly. Transfer to a 50 mL high-pressure reactor.

[0039] S2 and the mixture solution from step S1 are heated at 80–220 °C for 4–24 h. After cooling to room temperature, the mixture is centrifuged at 12000 rpm / min for 10 min, the supernatant is collected, filtered through a 0.22 μm filter membrane, the filtrate is freeze-dried, and then washed with solvent. The washing liquid is then evaporated using a rotary evaporator to remove the solvent and obtain dried carbon dots.

[0040] S3. Add an appropriate amount of water to the carbon dots obtained in step S2 to obtain a 2 mg / mL carbon dot aqueous solution, and adjust the pH of the solution with 5 M sodium hydroxide.

[0041] S4. Take 1000 volume parts of the solution from step S3, add 50-800 volume parts of 1M copper nitrate, shake well to obtain a sky blue hydrogel, centrifuge the hydrogel at 12000 rpm / min for 10 min, discard the supernatant, and wash three times with ultrapure water to obtain a pure copper cross-linked carbon dot hydrogel nanozyme.

[0042] (1) The amount of 1,2,3,4-butanetetracarboxylic acid, reaction temperature and time, and the amount of copper nitrate.

[0043] The results showed that with increasing dosage of 1,2,3,4-butanetetracarboxylic acid, the catalytic and gelling abilities gradually increased and eventually reached equilibrium, with the optimal dosage being 281 mg; with increasing reaction temperature, the catalytic and gelling abilities gradually increased and eventually reached equilibrium, with the optimal reaction temperature being 200 °C; with increasing reaction time, the catalytic and gelling abilities gradually increased and eventually reached equilibrium, with the optimal reaction time being 12 h; and with increasing dosage of copper nitrate, the gelling ability gradually increased and eventually decreased, with the optimal dosage of copper nitrate being 100 μL.

[0044] (2) Preparation of copper-crosslinked carbon dot hydrogel nanozymes under optimal conditions:

[0045] 315.2 mg of citric acid, 281 mg of 1,2,3,4-butanetetracarboxylic acid, and 20 mL of ultrapure water were added sequentially to a container and sonicated for 5 min to mix thoroughly. The mixture was then transferred to a 50 mL high-pressure reactor and heated at 200 °C for 12 h. After cooling to room temperature, the mixture was centrifuged at 12000 rpm for 10 min, and the supernatant was collected and filtered through a 0.22 μm filter membrane. The filtrate was freeze-dried, washed with acetonitrile, and the acetonitrile was removed by rotary evaporation to obtain dried carbon dots. An appropriate amount of water was added to prepare a 2 mg / mL carbon dot aqueous solution, and the pH of the solution was adjusted to 7 with 5 M sodium hydroxide. 1000 μL of the 2 mg / mL carbon dot solution was taken, and 100 μL of 1 M copper nitrate was added. The mixture was shaken well to obtain a sky-blue hydrogel. The hydrogel was centrifuged at 12000 rpm for 10 min, the supernatant was discarded, and the mixture was washed three times with ultrapure water to obtain a pure copper-crosslinked carbon dot hydrogel nanozyme.

[0046] Figure 1 This is a photograph of a copper-crosslinked carbon dot hydrogel nanozyme; the sky-blue adhesive can be clearly seen firmly adhering to the bottom of the bottle. From... Figure 2As can be seen, dopamine (DA) can only be catalyzed when copper-crosslinked carbon dot hydrogel nanozymes and H2O2 are present simultaneously, which indicates that copper-crosslinked carbon dot hydrogel nanozymes (BC-CDs@Cu) have strong peroxidase catalytic properties.

[0047] Example 2: Application of copper cross-linked carbon dot hydrogel nanozyme in the detection of tert-butylhydroquinone

[0048] (1) Construction of the colorimetric sensor: 100 μL of phosphate buffer solution (0.2 M, pH 7), 200 μL of BC-CDs@Cu (4 mg / mL), 10 μL of TBHQ solution of different concentrations, 100 μL of dopamine (20 mM), and 60 μL of H2O2 (100 mM) were added, and water was added to dilute to a total volume of 2 mL. The mixture was thoroughly mixed and reacted at room temperature for 10 min. The UV-Vis absorption spectra were measured, and TBHQ was detected based on the relationship between the absorption peak intensities of the dopamine oxidation product (aminochrome) at 480 nm corresponding to different concentrations of TBHQ. Figure 3 As shown, with the increase of TBHQ concentration, the absorption peak intensity of aminochrome at 480 nm gradually weakens. The TBHQ concentration and the absorbance of aminochrome show a linear relationship in the range of 0.5-20 μM, with a detection limit of 290 nm.

[0049] (2) Visualization Sensor Construction: 10 μL of phosphate buffer solution (0.2 M, pH 7), 160 μL of BC-CDs@Cu, 20 μL of TBHQ solution at different concentrations, 5 μL of dopamine at 40 mM, and 6 μL of H2O2 at 100 mM were added sequentially to a 96-well microplate. The mixtures were thoroughly mixed, and the reaction was allowed to proceed at room temperature for 10 min. Color changes in each well were recorded using a smartphone, and the RGB values ​​of the images were analyzed using ImageJ image processing software. Figure 4 As shown, the color of BC-CDs@Cu exhibits a gradient change with increasing TBHQ concentration, and the B / R value increases with increasing TBHQ concentration. The TBHQ concentration and B / R value show a linear relationship in the range of 5-100 μM, with a detection limit of 1.1 μM.

[0050] The above description merely illustrates preferred embodiments of the present invention, and while the description is relatively specific and detailed, it should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications, improvements, and substitutions without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this patent should be determined by the appended claims.

Claims

1. A method for preparing copper-crosslinked carbon dot hydrogel nanozymes, characterized in that: This method uses 1,2,3,4-butanetetracarboxylic acid and citric acid as raw materials to prepare carbon dots in one step via a hydrothermal method, and then uses Cu... 2+ Copper-crosslinked carbon dot hydrogel nanozymes were obtained using these as catalytic centers. The method includes the following steps: S1. Add citric acid, 1,2,3,4-butanetetracarboxylic acid and ultrapure water to a container in sequence, and sonicate for a period of time to mix evenly to obtain a mixture solution. S2. Transfer the mixture solution obtained in step S1 to a high-pressure reactor and heat it to react. After cooling to room temperature, centrifuge it at high speed, collect the supernatant, filter it through a filter membrane, and freeze-dry, wash with solvent, and remove the solvent by rotary evaporation to obtain dried carbon dots. S3. Add water to the carbon dots obtained in step S2 to prepare an aqueous solution of carbon dots, and adjust the pH value of the solution with sodium hydroxide. S4. Take the carbon dot aqueous solution and copper nitrate solution obtained in step S3 and mix them evenly to obtain a sky blue hydrogel. After centrifuging the hydrogel at high speed, take the precipitate and wash the precipitate several times with ultrapure water to obtain pure copper cross-linked carbon dot hydrogel nanozyme.

2. The method for preparing copper cross-linked carbon dot hydrogel nanozymes according to claim 1, characterized in that: In step S1, 315.2 parts by weight of citric acid and 70.2-702 parts by weight of 1,2,3,4-butanetetracarboxylic acid are added; in step S2, the reaction temperature is heated to 80-220 °C, the reaction time is 4-24 h, and the pore size of the filter membrane is 0.22 μm; in step S3, the pH of the solution is adjusted to 7 using 5 M sodium hydroxide; in step S4, 1000 parts by volume of carbon point aqueous solution and 50-800 parts by volume of copper nitrate solution are added.

3. The method for preparing copper cross-linked carbon dot hydrogel nanozymes according to claim 2, characterized in that: In step S1, the amount of 1,2,3,4-butanetetracarboxylic acid used is 281 parts by weight; the ultrasonic time is 5 min; in step S2, the heating reaction temperature is 200 ℃; the heating reaction time is 12 h; the solvent is acetonitrile; and in step S4, the amount of copper nitrate solution used is 100 parts by volume.

4. A copper cross-linked carbon dot hydrogel nanozyme prepared by the preparation method according to any one of claims 1 to 3.

5. The application of a copper-crosslinked carbon dot hydrogel nanozyme prepared by the preparation method according to any one of claims 1 to 3, characterized in that: A colorimetric and visualization sensor was constructed based on the copper-crosslinked carbon dot hydrogel nanozyme for the detection of tert-butylhydroquinone (TBHQ).

6. The application according to claim 5, characterized in that, The method for detecting tert-butylhydroquinone based on a colorimetric sensor constructed using the copper-crosslinked carbon dot hydrogel nanozyme in the application includes: (1.1) Preparation of multiple detection solutions: Phosphate buffer solution, copper cross-linked carbon dot hydrogel nanozyme, tert-butylhydroquinone solution, dopamine, H2O2 were mixed and diluted with water. After mixing evenly, the mixture was reacted at room temperature. Multiple detection solutions were obtained by adding different concentrations of tert-butylhydroquinone solution. (1.2) After reacting the detection solution obtained in step (1.1) at room temperature for 10 min, the ultraviolet-visible absorption spectrum was measured; (1.3) tert-butylhydroquinone was detected based on the relationship between the absorption peak intensities of dopamine oxidation products at 480 nm corresponding to different concentrations of TBHQ; Alternatively, the method for detecting tert-butylhydroquinone based on a visual sensor constructed using the copper-crosslinked carbon dot hydrogel nanozyme in the application includes: (2.1) Preparation of multiple test samples: Phosphate buffer solution, copper cross-linked carbon dot hydrogel nanozyme, tert-butylhydroquinone solution, dopamine, and H2O2 were mixed evenly. Multiple test solutions were obtained by adding different concentrations of tert-butylhydroquinone solution. Each test solution was added to a 96-well plate and reacted at room temperature for 10 min to obtain multiple test samples. (2.2) Use a camera to record images of the color of each well of the 96-well microplate; (2.3) The RGB values ​​of the color image obtained in step (2.2) are analyzed by image processing software to obtain the linear equation between TBHQ concentration and RGB values, thereby achieving the purpose of colorimetric detection of TBHQ.

7. The application according to claim 6, characterized in that, In step (1.1), the volume ratio of each component in the detection solution is as follows: 200 parts by volume of copper cross-linked carbon dot hydrogel nanozyme, 100 parts by volume of phosphate buffer solution, 10 parts by volume of TBHQ solution, 100 parts by volume of dopamine, and 60 parts by volume of H2O2; diluted with water to a total volume of 2000 parts by volume; the concentration of the copper cross-linked carbon dot hydrogel nanozyme is 4 mg / mL; the concentration of the phosphate buffer solution is 0.2 M with a pH of 7; the concentration of dopamine is 20 mM; and the concentration of H2O2 is 100 mM.

8. The application according to claim 6, characterized in that: In step (2.1), the volume ratio of each part in the detection solution is as follows: 10 parts phosphate buffer solution, 160 parts copper cross-linked carbon dot hydrogel nanozyme, 20 parts TBHQ solution, 5 parts dopamine, and 6 parts H2O2; the concentration of phosphate buffer solution is 0.2 M and the pH value is 7; the concentration of dopamine is 40 mM and the concentration of H2O2 is 100 mM.

9. The application according to any one of claims 6 to 8, characterized in that: In step (1.3), as the concentration of TBHQ increases, the absorption peak intensity of the dopamine oxidation product at 480 nm gradually decreases. The concentration of TBHQ and the absorbance of the dopamine oxidation product show a linear relationship in the range of 0.5-20 μM, with a detection limit of 290 nM. In step (2.3), as the concentration of TBHQ increases, the color of the copper cross-linked carbon dot hydrogel nanozyme shows a gradient change, the B / R value increases with the concentration of tert-butylhydroquinone, the TBHQ concentration and the B / R value show a linear relationship in the range of 5-100 μM, and the detection limit is 1.1 μM.

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