Application of hypericin-derived carbon dots in colorimetric detection of total antioxidant capacity

Abstract

The application discloses application of ariophyllum viceroy flower derived carbon dots in colorimetric detection of total antioxidant capacity (TAC). The application takes ariophyllum viceroy flower as a precursor, and prepares carbon dots (CDs) with light response oxidase activity through one-step hydrothermal method. The catalytic activity of the CDs follows the Michaelis equation. The prepared CDs can catalyze colorless 3,3',5,5T-tetramethylbenzidine (TMB) to be oxidized into blue oxTMB under ultraviolet light irradiation, and generate characteristic ultraviolet absorption. Based on the reduction of antioxidants on oxTMB, the ultraviolet absorbance is reduced, and the solution color is lightened, and a colorimetric detection TAC platform is constructed. The platform takes ascorbic acid as a model, has a good linear response in the range of 0.05-10.0 muM of the concentration of the ascorbic acid, has anti-interference ability on common ions and amino acids, and is successfully applied to detection of TAC in actual samples such as tomatoes, grapefruits and strawberries. The method has the advantages of green raw materials, simple preparation, convenient operation and high sensitivity, and has wide application prospect in the fields of food analysis and biological detection.

Description

Technical Field

[0001] This invention relates to the field of colorimetric detection of carbon dots, and specifically to the application of Hypericum carbon dots in the colorimetric detection of total antioxidant capacity. Background Technology

[0002] Free radicals produced during human metabolism are not only key mediators for the immune system to kill pathogens, but also participate in intercellular signal transduction. Among them, hydroxyl radicals (·OH) and superoxide anions (·O2) are particularly important. - ) and singlet oxygen ( 1 Oxygen radicals such as O2 are typical reactive oxygen species (ROS). However, excessive ROS production can induce oxidative stress damage, significantly increasing the risk of inflammation, organ damage, cancer, cardiovascular disease, and neurodegenerative diseases.

[0003] Antioxidants effectively neutralize excess reactive oxygen species (ROS), playing an indispensable role in maintaining redox homeostasis, protecting cells from oxidative damage, and reducing disease risk. While endogenous antioxidants in the body (such as glutathione and superoxide dismutase, SOD) possess scavenging capabilities, their effectiveness is limited. Exogenous antioxidants, such as dietary sources of vitamin C (ascorbic acid) and vitamin E, as well as polyphenols found in tea and berries, can effectively compensate for deficiencies in endogenous defenses. Therefore, accurately assessing the antioxidant content in the diet and implementing targeted dietary adjustments is of significant practical importance for promoting public health.

[0004] Total antioxidant capacity (TAC) is defined as a comprehensive antioxidant index used to assess free radical scavenging activity. Various methods have been developed for TAC determination, primarily including high-performance liquid chromatography (HPLC), electrochemical analysis, chemiluminescence, and spectrometry. Colorimetry, as a type of spectrometry, is based on the in-situ visible colorimetric reaction of the substrate under catalysis, following the Lambert-Beer law. Typically, colorless 3,3′,5,5′-tetramethylbenzidine (TMB) can be oxidized to a blue complex (oxTMB) with a characteristic absorption spectrum, and is therefore widely used as a universal chromogenic substrate in colorimetric analysis. Activated oxygen or hydrogen peroxide catalyzes the chemical reaction, generating reactive oxygen species (ROS), which further promotes substrate oxidation. Based on the inhibitory effect of antioxidants on the oxidation reaction, ROS is reduced, leading to color changes and variations in the spectral signal. TAC can then be determined by quantifying these measurement signals in the presence or absence of antioxidants.

[0005] Nanozymes have been widely used in the construction of colorimetric sensors due to their advantages such as high catalytic efficiency, good stability, economy, and large-scale preparation. Carbon dots, as an emerging type of carbon-based nanozyme, have attracted widespread attention due to their simple preparation, good water solubility, and high biocompatibility. To improve catalytic performance, most carbon dots reported for TAC detection are currently doped with metal elements (e.g., Fe-adsorbed carbon dots) (Li, J., et al. Fluorescence / colorimetric intelligent sensor based on Fe-adsorbing carbon dots nanozymes for selective recognition and quantification of total antioxidant capacity in foods and cells. Food Chemistry c(2025), 490:145052), or prepared using artificially synthesized chemical reagents (e.g., citric acid, aniline, urea, etc.) as precursors (Li, J., et al. Carbon dots as light-responsive oxidase-like nanozyme for colorimetric detection of total antioxidant capacity in fruits. Food Chemistry(2023), 405:134749-134755.). While these metal-assisted or chemically derived carbon dots enhance activity to some extent, they also bring problems such as metal leakage, chemical pollution, environmental toxicity, and decreased biocompatibility, limiting their further application in the field of bioassay.

[0006] Therefore, to avoid contamination and toxic side effects from metals and chemical reagents, this invention studies a colorimetric detection method for TAC using undoped biomass carbon dots with oxidase activity. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the purpose of this invention is to provide an application of Hypericum perforatum carbon dots in the colorimetric detection of total antioxidant capacity (TAC). This invention uses Hypericum perforatum as the sole precursor and prepares a pure biomass carbon dot with good water solubility, low toxicity, and no metals through a one-step hydrothermal method. This carbon dot exhibits photoresponsive oxidase-like properties and can be used for TAC colorimetric detection.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] The preparation of a Hypericum-derived carbon dot includes the following steps:

[0010] (1) Mix Hypericum flowers, water or ethanol or a mixture of both at a mass-volume ratio of 1:(1-50) and place them in a hydrothermal reactor. Perform the hydrothermal reaction at 120-230°C for 5-20 hours.

[0011] (2) Centrifuge the product obtained in step (1) to remove insoluble matter, filter it with a 0.22 μm filter membrane, and then dialyze it with a dialysis bag for 12-48 hours to remove impurities, and obtain carbon dot solution;

[0012] (3) Freeze-dry the carbon dot solution obtained in step (2) to obtain carbon dot solid.

[0013] The carbon dots have a particle size range of 2-10 nm and contain C, N, O elements and various related functional groups.

[0014] This invention provides an application of Hypericum-derived carbon dots in the colorimetric determination of total antioxidant capacity, the specific steps of which are as follows:

[0015] (1) The carbon dots were mixed with 3,3′,5,5′-tetramethylbenzidine (TMB) in an acidic buffer solution to form a CDs / TMB system. The system underwent an oxidation reaction under ultraviolet light to generate a blue oxidation product oxTMB. The absorbance of the system was measured.

[0016] (2) Add different concentrations of antioxidants to the above reaction system. Based on the reduction effect of antioxidants on oxTMB, oxTMB is reduced and the absorbance of the system decreases. Through its absorbance, establish the linear relationship between absorbance and antioxidant concentration.

[0017] (3) The treated actual sample was added to the CDs / TMB system after UV irradiation, the absorbance was measured, and the content of antioxidants in the sample was calculated and the TAC level was evaluated based on the above linear relationship.

[0018] Optionally, the ultraviolet light wavelength is 365nm and the irradiation time is 5 to 30 minutes;

[0019] Preferably, the ultraviolet light irradiation time is 20 minutes;

[0020] Optionally, the acidic solution is an HAc-NaAc buffer solution with a concentration of 0.1-0.2M and a pH value of 3.0-6.0;

[0021] Preferably, the HAc-NaAc buffer solution has a concentration of 0.2M and a pH value of 3.5;

[0022] Optionally, the concentration of the carbon dots in the catalytic system is 10.0–30.0 μg / mL;

[0023] Preferably, the concentration of the carbon dots in the catalytic system is 15 μg / mL;

[0024] Optionally, the concentration of TMB in the catalytic system is 1.0–5.0 mM;

[0025] Preferably, the concentration of TMB in the catalytic system is 2.5 mM;

[0026] Optionally, the maximum absorption wavelength in the ultraviolet absorption characteristics is located in the range of 650–655 nm;

[0027] Preferably, the maximum absorption wavelength in the ultraviolet absorption characteristic is located at 652 nm.

[0028] The linear relationship between absorbance and antioxidant concentration was established using ascorbic acid as a model, with concentrations of 0, 0.05, 0.1, 0.25, 0.5, 0.75, 1.0, 1.5, 2.0, 2.5, 5.0, 7.5, 10.0, and 12.5 μM. The absorbance and ascorbic acid concentration showed a linear relationship in the range of 0.05–10.0 μM, and the standard curve was y = -0.0379x + 0.392(R). 2 =0.996), where y is the absorbance at 652 nm and x is the concentration of ascorbic acid, and the limit of detection is 0.043 μM (S / N = 3).

[0029] The TAC detection method provided by this invention is effective for common salt ions (Na+). + K + Cu 2+ Ca 2+ Zn 2+ ) and amino acids (L-Arg, D-Ala, Sar, L-His) have good selectivity and anti-interference ability.

[0030] The present invention has the following beneficial technical effects:

[0031] (1) The St. John's wort-derived carbon dots provided by this invention are prepared by a one-step method, without metal doping, without the use of toxic reagents, and are green, environmentally friendly and inexpensive.

[0032] (2) The Hypericum-derived carbon dots provided by the present invention are a photoresponsive oxidase, and their catalytic process does not require the use of the hazardous chemical H2O2.

[0033] (3) The colorimetric method for detecting TAC provided by the present invention does not require waiting for the reaction process, is visual, easy to operate, highly accurate, and has good selectivity and anti-interference ability. Detailed Implementation

[0034] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Attached Figure Description

[0035] Figure 1 TEM image of carbon dots in Hypericum perforatum;

[0036] Figure 2 FTIR image of carbon dots in Hypericum perforatum;

[0037] Figure 3 XPS image of St. John's wort carbon dots;

[0038] Figure 4 High-resolution XPS images of carbon dots in Hypericum (A: C1s spectrum; B: N1s spectrum);

[0039] Figure 5 Absorption spectra of different systems with and without UV irradiation: TMB (a, b), CDs (c, d) and CDs+TMB (e, f);

[0040] Figure 6 Photoresponse properties of Hypericum carbon dots – light control experiment;

[0041] Figure 7 Activity verification and catalytic mechanism of carbon dots in Hypericum perforatum: Free radical capture experiment;

[0042] Figure 8 Linearity of Hypericum carbon dots for colorimetric detection of TAC: absorption spectra of a series of ascorbic acid concentrations (A), standard curve of absorbance against ascorbic acid concentration in the range of 0.05–10.0 μM (B), and visualization photographs of the solutions (C).

[0043] Figure 9 Comparison of selectivity (A) and anti-interference (B) of Hypericum perforatum carbon dots for colorimetric detection of TAC.

[0044] Example 1: Preparation and Characterization of Hypericum Carbon Dots

[0045] Preparation: Hypericum carbon dots (CDs) were prepared using a one-step hydrothermal method with Hypericum flowers as the carbon source. 5.0 g of washed and dried Hypericum flowers and 20 mL of ethanol were added to a polytetrafluoroethylene-lined high-pressure reactor and reacted at 180 °C for 10 h. After natural cooling, the solid residue was removed by filtration. The resulting brownish-brown colloidal solution was purified using a dialysis bag (molecular weight cutoff 1000 Da) for 48 h, and finally freeze-dried to obtain brownish-black powder CDs.

[0046] Characterization: Morphology was observed using HR-TEM. The CDs were quasi-spherical, uniformly dispersed, and their particle size distribution ranged from 2 to 10 nm. Figure 1 FTIR spectroscopy Figure 2 The display shows 3430cm -1 The broad peak at 1595 cm⁻¹ is due to the OH stretching vibration. -1 The peak is attributed to the C=O / NH stretching vibration, 1357cm. -1 1159cm -1 and 770cm -1 The corresponding stretching vibrations for CO / CN, CC, and CH indicate the presence of hydrophilic groups such as hydroxyl, carbonyl, and amino groups on the surface. XPS full spectrum ( Figure 3 The presence of C, N, and O was confirmed, with atomic percentages of 82.6 at%, 1.1 at%, and 12.3 at%, respectively. High-resolution C1s and N1s spectra (…) Figure 4 The results were fitted to three peaks at 284.0 eV (CC), 285.6 eV (CO / CN), and 287.5 eV (C=O), and three peaks at 397.7 eV (CNC), 399.3 eV (CNH), and 400.8 eV ((C)3-N), respectively. The XPS results were corroborated by FTIR, confirming that the CDs surface contains abundant oxygen- and nitrogen-containing functional groups.

[0047] Example 2: CDs photoresponsive oxidase activity test

[0048] The oxidase-like activity of CDs was determined using TMB as a chromogenic substrate. The CDs / TMB+ irradiation system consisted of 100 μL CDs (0.3 mg / mL), 200 μL TMB (25 mM), and 1700 μL HAc-NaAc buffer (0.2 M, pH 3.5), with a total volume of 2 mL, and was irradiated under 365 nm UV light for 20 min. Correspondingly, five additional control experiments were set up: TMB+ alone with / without light, CDs alone with / without light, and CDs / TMB+ without light. The absorption spectra of these systems were measured in the range of 200-800 nm, and the absorbance at 652 nm was recorded. The results are as follows: Figure 5 As shown, the CDs / TMB+ illumination system has a characteristic absorption peak at 652 nm, while other solutions show no color change and no obvious ultraviolet absorption.

[0049] Example 3: Photoresponse Performance – Light Control Experiment

[0050] The light-controlled test system was prepared as follows: 100 μL CDs (0.3 mg / mL), 200 μL TMB (25 mM), and 1700 μL HAc-NaAe buffer (0.2 M, pH 3.5). The 365 nm UV lamp was alternately switched on and off every 2 minutes, and the absorbance change was observed within 0-50 minutes to verify the controllability of UV irradiation on the oxidation process. The results are as follows: Figure 6 As shown, the absorbance increased significantly after UV irradiation (light on mode), while there was no significant change in absorbance after the UV lamp was turned off (light off mode), indicating that the oxidase activity of CDs depends on UV irradiation.

[0051] Example 4 Activity Verification and Catalytic Mechanism – Free Radical Scavenging Experiment

[0052] Reactive oxygen species were captured using quenchers tryptophan (Try), hydroxylamine hydrochloride (HAH), tert-butanol (TBA), superoxide dismutase (SOD), and ethylenediaminetetraacetic acid (EDTA).

[0053] First, prepare the CDs / TMB solution: mix 100 μL CDs (0.3 mg / mL), 200 μL TMB (25 mM), and 1700 μL HAc-NaAc buffer (0.2 M, pH 3.5), and irradiate under 365 nm UV light for 20 min. Then, introduce these quenchers into the above solution. Figure 7 As shown, the ratio of the UV absorbance of the solution at 652 nm after adding the quencher to the absorbance before adding the quencher is defined as the relative activity of CDs. Figure 7 It can be seen that Try caused a slight decrease in absorbance, while HAH, TBA, and SOD caused a significant decrease. Therefore, it can be inferred that ·OH and ·O2 were generated during the catalytic process. - .

[0054] Example 5: Construction of linear relationship for colorimetric detection of TAC

[0055] Hypericum carbon dots were used in the colorimetric detection of TAC, and a linear relationship was established using ascorbic acid as a model. A basal solution was prepared containing CDs (15 μg / mL), TMB (2.5 mM), and HAc-NaAc buffer (0.2 M, pH 3.5), and irradiated under UV light (365 nm) for 20 min. A series of AA concentrations (final concentrations of 0, 0.05, 0.1, 0.25, 0.5, 0.75, 1.0, 1.5, 2.0, 2.5, 5.0, 7.5, 10.0, and 12.5 μM) were added to each basal solution to a total volume of 2.0 mL. After reacting for 5 min, the absorbance at 652 nm was measured, and a curve was plotted against the AA concentration. Results ( Figure 8It was found that absorbance and ascorbic acid concentration showed a linear relationship in the range of 0.05–10.0 μM, and the standard curve was y = -0.0379x + 0.392(R). 2 =0.996), where y is the absorbance at 652 nm and x is the concentration of ascorbic acid, and the limit of detection is 0.043 μM (S / N = 3).

[0056] Example 6: Selectivity and Anti-interference

[0057] Prepare the basic solution: containing CDs (15 μg / mL), TMB (2.5 mM), and HAc-NaAc buffer (0.2 M, pH 3.5), and irradiate under ultraviolet light (365 nm) for 20 min.

[0058] Selectivity experiment: Add buffer (as blank), ascorbic acid (final concentration 5.0 μM), or interferon (Na+) to 12 aliquots of the above basic solution. + K + Cu 2+ Ca 2+ Zn 2+ L-arginine (L-Arg), D-alanine (D-Ala), sarcosine (Sar), L-cysteine ​​(L-Cys), L-histidine (L-His) (final concentration 5.0 μM), with the total volume kept constant;

[0059] Similarly, in the anti-interference experiment, the concentration of ascorbic acid was 5.0 μM and the concentration of the interfering substance was 25.0 μM, and both were added simultaneously.

[0060] The results showed that, except for L-Cys which caused some interference due to its own reducing properties, the other substances had minimal impact on the absorbance of the AA detector, indicating that there was essentially no interference. Figure 9 ).

[0061] Application Example 1: Detection of actual samples

[0062] Three types of fruit and vegetable samples—tomato, grapefruit, and strawberry—were selected as actual samples. A spiked recovery method was used to verify the applicability of the constructed platform. These samples were washed and juiced, and the juice was filtered through a 0.22 μm filter membrane for later use. Simultaneously, a basic solution (containing CDs (15 μg / mL), TMB (2.5 mM), and HAc-NaAc buffer (0.2 M, pH 3.5)) was prepared and irradiated under UV light (365 nm) for 20 min.

[0063] Blank: Take 1990 μL of the above basic solution and add 10 μL of buffer solution to it.

[0064] Control: Take 1990 μL of the above basic solution and add 5 μL of ascorbic acid (0.25 mM) and 5 μL of buffer solution.

[0065] Test: Take 1990 μL of the above basic solution, add 5 μL of the treated actual sample (the grapefruit sample was diluted with ultrapure water by one-fold) and 5 μL of buffer solution.

[0066] Spiked sample: Take 1990 μL of the above basic solution, add 5 μL of the treated actual sample (the grapefruit sample was diluted with ultrapure water by one-fold) and 5 μL of ascorbic acid (0.25 mM).

[0067] The absorbance of the above solution at 652 nm was measured in triplicate, and the results were substituted into the linear equation to calculate the concentration of antioxidant in the actual sample. The total antioxidant capacity (calculated as ascorbic acid equivalents C1, C2, and C3) and ascorbic acid recovery rate in the original sample are shown in Table 1.

[0068] Table 1

[0069]

Claims

1. An application of Hypericum-derived carbon dots in the colorimetric determination of total antioxidant capacity (TAC), characterized in that, Includes the following steps: (1) Carbon dots (CDs) were prepared and purified by a one-step hydrothermal method using Hypericum as a precursor; (2) The carbon dots (CDs) were mixed with 3,3′,5,5′-tetramethylbenzidine (TMB) in an acidic buffer solution to form a CDs / TMB system. The system underwent an oxidation reaction under ultraviolet light to generate a blue oxidized product oxTMB. The absorbance of the system was measured. (3) Add different concentrations of antioxidants to the above reaction system. Based on the reduction effect of antioxidants on oxTMB, oxTMB is reduced and the absorbance of the system decreases. By measuring its absorbance, the linear relationship between absorbance and antioxidant concentration is established. (4) The treated actual sample was added to the CDs / TMB system after UV irradiation, the absorbance was measured, and the content of antioxidants in the sample was calculated by substituting it into the above linear relationship, and the TAC level was evaluated.

2. The method according to claim 1, characterized in that, The concentration of CDs in the catalytic system is 10.0–30.0 μg / mL; the concentration of TMB is 1.0–5.0 mM; the acidic buffer solution is an acetate-sodium acetate buffer solution (HAc-NaAc) with a concentration of 0.1–0.2 M and a pH of 3.0–6.0; the ultraviolet light irradiation wavelength is 365 nm and the irradiation time is 5–30 min.

3. The method according to claim 1, characterized in that, The maximum absorption wavelength for absorbance measurement is 650–655 nm.

4. The method according to claim 1, characterized in that, The colorimetric platform for TAC detection uses ascorbic acid as a model, establishing a linear relationship between absorbance and ascorbic acid concentration in the range of 0.05–10.0 μM. The linear equation is y = -0.0379x + 0.392(R). 2 =0.996), where y is the absorbance at 652 nm and x is the concentration of ascorbic acid; the TAC level in the actual sample is expressed as ascorbic acid equivalent.

5. A colorimetric reagent kit for detecting TAC, characterized in that, It includes the carbon dots, TMB, HAc-NaAc buffer, and ultraviolet irradiation conditions as described in any one of claims 1-4.

Images