Preparation method and application of light-regulated dual-analogous enzyme bimetallic carbon-based hybrid

Abstract

The application discloses a preparation method and application of a light-regulated dual-analogous enzyme bimetallic carbon-based hybrid. Two of lanthanum chloride, ytterbium chloride and gadolinium chloride aqueous solution are added into a PVP ethylene glycol solution, glutathione is used as a reducing agent, and oil bath reaction is carried out, so that a bimetallic carbon-based hybrid Yb-Gd-CDs, La-Yb-CDs or La-Gd-CDs which is light-regulated and simultaneously has peroxide mimic enzyme and oxide mimic enzyme activity is prepared. The bimetallic carbon-based hybrid can be used as a peroxide mimic enzyme to detect D-proline content and D-alanine content, can be used as an oxide mimic enzyme to detect antioxidant small molecule content and total antioxidant capacity of cell lysate, and can be used as an oxide mimic enzyme to detect total antioxidant capacity of fruits and fruit juice, so that rapid and accurate detection of target to-be-detected substances can be realized.

Description

Technical Field

[0001] This invention relates to the fields of chemical and biological sensing and biological detection, specifically to a method for preparing and applying a photo-regulated dual-mimicryse bimetallic carbon-based hybrid. Background Technology

[0002] Peroxidases, as one of the most effective biocatalysts, possess high catalytic activity and substrate specificity, playing a crucial role in medicine, industry, agriculture, and biology. However, inherent limitations of natural enzymes, such as high preparation and purification costs, low operational stability, high environmental sensitivity, and difficulties in recycling and reuse, restrict their further applications. Based on this situation, various artificial enzyme mimics have been rapidly explored and developed. Peroxidase mimics are typically organic or inorganic molecules with peroxidase-like activities, exhibiting similar substrate selectivity and high catalytic activity. Peroxidase mimics are widely used in biomedicine, environmental monitoring, and food safety. Compared to natural peroxidases, peroxidase mimics have many advantages, such as better thermal stability, broader substrate adaptability, and easier synthesis and regulation. Peroxidase mimics include nanomaterials, metal oxides, noble metals and metal-organic frameworks, porphyrins, biomolecules, metal complexes, and polymers. Many small molecules and ions have also been found to possess peroxidase mimic activity, providing new and promising materials for related research.

[0003] Oxygenases are catalysts that mimic natural enzymes, exhibiting similar catalytic activity. These mimics can be synthesized from organic or inorganic compounds to mimic the catalytic activity of natural enzymes, thus acting as catalysts in various chemical reactions. Oxygenases typically possess high catalytic efficiency and specificity, making them suitable for catalytic reactions in biomedicine, environmental protection, and industrial production. These mimics can be designed with specific structures to adapt to different reaction conditions and substrate types. In recent years, oxidases have received increasing attention in the field of catalysis and have been widely applied in catalytic reactions across various sectors.

[0004] Proline (Pro), an important amino acid, plays a wide range of physiological functions in organisms. It participates in various biological processes, including protein synthesis, cell proliferation and differentiation, and signal transduction. Furthermore, proline is widely used in drug development, such as as a target for anti-tumor drugs or as an antibacterial and antioxidant agent. Whether in microbial synthesis, biotransformation, stress response mechanisms, or in drug design and environmental science, proline plays a vital role. Meanwhile, the chemical properties of proline have attracted widespread attention from researchers, including its conformational analysis, electronic properties, and interactions with other molecules. Therefore, developing a simple, rapid, and reliable method for the detection of D-proline is of great significance.

[0005] Alanine is a very important amino acid, belonging to the non-essential amino acid group. In living organisms, D-alanine is relatively scarce, while L-alanine is the more common form, primarily involved in biochemical processes such as protein synthesis. D-alanine has wide-ranging research applications, playing a crucial role in drug synthesis and biomedical research, and can be used to synthesize specific classes of bioactive molecules, such as antibiotics and antiviral drugs. As a neurotransmitter, D-alanine plays a vital role in the functional regulation of the nervous system. It also plays an important role in protein synthesis and metabolism, particularly in protein nutrition and healthy diets. The detection of D-alanine is significant in medical diagnostics, food safety, and biochemical research. The application of biosensor technology in D-alanine detection is also increasing; by constructing specific biosensors, real-time monitoring and rapid detection of D-alanine can be achieved. In summary, research on D-alanine in various fields is continuously deepening, providing an important foundation for a better understanding of its biological functions and application value.

[0006] Antioxidant small molecules are small molecule compounds with antioxidant properties that help reduce or prevent oxidative stress and damage, thereby protecting cells and tissues in the body. Antioxidant small molecules exert their antioxidant effects by scavenging free radicals, neutralizing oxidants, or enhancing their own antioxidant systems. Common antioxidant small molecules include antioxidants (ascorbic acid, tartaric acid, and tannic acid), biothiols (GSH, Cys, and Hcy), and dopamine. These small molecules play an important role in protecting cells from oxidative damage and help maintain a healthy state. Antioxidant small molecules have wide applications in food, pharmaceuticals, and cosmetics.

[0007] Total Antioxidant Capacity (TAC) is an indicator used to assess the antioxidant capacity of an organism or biological sample. The TAC test measures a sample's overall responsiveness to oxidants in the presence of antioxidants, thus reflecting the sample's antioxidant capacity. It indirectly evaluates a sample's antioxidant capacity by assessing its ability to reduce specific oxidants or scavenge free radicals. TAC detection is of great significance in scientific research and clinical practice, helping to assess the body's antioxidant capacity and guiding dietary adjustments and drug treatments.

[0008] Our experiments demonstrate that the bimetallic carbon-based hybrids Yb-Gd-CDs, La-Yb-CDs, and La-Gd-CDs all possess dual-mimicry enzyme catalytic capabilities, exhibiting both peroxidase and oxidase mimicry abilities. In the presence of these bimetallic carbon-based hybrids, H2O2 is catalytically decomposed to generate hydroxyl radicals. These hydroxyl radicals oxidize TMB (3,3',5,5'-tetramethylbenzidine) to generate oxidized TMB (Ox-TMB). Different concentrations of oxidized TMB exhibit different colors, thus demonstrating their peroxidase mimicry properties. Meanwhile, in the absence of H2O2, the aforementioned bimetallic carbon-based hybrids can react with TMB to directly oxidize it to Ox-TMB, resulting in a colorimetric reaction, demonstrating their oxidase-mimicking properties. Furthermore, both of these bimetallic carbon-based hybrid dual-mimicking enzymes exhibit photosensitivity. Their peroxidase-mimicking properties effectively oxidize TMB in both light and dark environments, with slightly stronger color development under light. Their oxidase-mimicking properties show significantly stronger TMB oxidation color development under light than in the dark, while the color development under dark is weaker. Based on this, we have established a light-controlled dual-mimicking enzyme detection method. This method can utilize the peroxidase-mimicking properties in the dark for quantitative detection of analytes, and also utilize the oxidase-mimicking properties under light for quantitative detection of analyte content. By light-controlled regulation, the mutual interference between the two mimicking enzymes in detecting samples is reduced, achieving accurate detection of different analytes. Summary of the Invention

[0009] To address the technical problem that current peroxide mimics or oxide mimics cannot meet the needs of practical applications, this invention provides a method for preparing and applying a photo-regulated dual mimic bimetallic carbon-based hybrid.

[0010] The present invention provides a method for preparing a photo-regulated dual-mimicry enzyme bimetallic carbon-based hybrid, wherein the photo-regulated dual-mimicry enzyme bimetallic carbon-based hybrid includes Yb-Gd-CDs, La-Yb-CDs, or La-Gd-CDs, and specifically includes the following steps:

[0011] S1. Dissolve PVP in ethylene glycol and stir until fully dissolved to obtain a PVP ethylene glycol solution;

[0012] S2. Add two of the following to the PVP glycol solution obtained in S1: ytterbium chloride aqueous solution, lanthanum chloride aqueous solution, and gadolinium chloride aqueous solution. Add the two solutions simultaneously, or add one and disperse it evenly before adding the other.

[0013] S3. Under stirring conditions, add HNO3 and / or acetic acid to the solution obtained in S2 to adjust the pH of the solution (adjust the pH to 4.5~5.0, preferably 4.8), then add glutathione solution and stir evenly. The molar ratio of glutathione to bimetal (lanthanum-ytterbium or ytterbium-gadolinium or lanthanum-gadolinium) is 1.1~1.3:0.9~1.1.

[0014] S4. The solution obtained in S3 is reacted with magnetic stirring at high temperature in an oil bath. After cooling, pale yellow light-regulated dual-mimicryse bimetallic carbon-based hybrids Yb-Gd-CDs, La-Yb-CDs, or La-Gd-CDs with blue-green fluorescence are obtained.

[0015] Furthermore, in S1, the ratio of polyvinylpyrrolidone (PVP) to ethylene glycol is 30~50 mg: 3.0~5.0 mL.

[0016] Furthermore, in S2, the amounts of the two solutions added are equal, and the concentration of the ytterbium chloride aqueous solution, lanthanum chloride aqueous solution, or gadolinium chloride aqueous solution is 10~30 mmol / L, more preferably 20 mmol / L.

[0017] Furthermore, in S3, the concentration of glutathione is 80~120 mmol / L, more preferably 100 mmol / L.

[0018] Furthermore, in S4, the reaction temperature of the oil bath is 100~140℃, more preferably 120℃; the reaction time is 5~10h. The optimal reaction time varies depending on the product. For example, the magnetic stirring reaction time for preparing Yb-Gd-CDs is 9h, and the magnetic stirring reaction time for preparing La-Yb-CDs is 6h.

[0019] The application of the photo-regulated dual-mimicryse bimetallic carbon-based hybrid obtained by the above preparation method as a peroxide mimicry in the detection of D-proline (D-Pro) content specifically includes the following steps: First, D-proline (D-Pro) is mixed with D-amino acid oxidase (DAAO) and reacted for 2-4 h (preferably 2.5 h). Then, Yb-Gd-CDs, La-Yb-CDs or La-Gd-CDs solutions, 3,3',5,5'-tetramethylbenzidine (TMB) solution, and acetate-sodium acetate buffer are added to the system and mixed well. The mixture is then reacted in a water bath for 2-4 h (preferably 2.5 h). Due to the different concentrations of H2O2 generated by D-amino acid oxidase catalyzing D-proline, the hydroxyl radicals (•OH) generated by Yb-Gd-CDs, La-Yb-CDs, or La-Gd-CDs catalyzing H2O2 exhibit different degrees of oxidation color development of TMB, i.e., the color of Ox-TMB is different. The content of D-proline in the sample is then calculated based on the standard curve of the absorbance value of Ox-TMB at 652 nm and the concentration of D-proline.

[0020] Further, the volume ratio of D-proline to D-amino acid oxidase is 1:0.9~1.1, the concentration of D-proline is 50µmol / L~10mmol / L, the concentration of D-amino acid oxidase is 0.04~0.06mg / mL (preferably 0.05mg / mL), and the water bath reaction temperature is 35~40℃ (preferably 37℃); the concentration of Yb-Gd-CDs, La-Yb-CDs or La-Gd-CDs solution is 6~7. The concentration of 3,3',5,5'-tetramethylbenzidine (TMB) is 4-6 mmol / L (preferably 5 mmol / L), the concentration of acetate-sodium acetate buffer is 0.1-0.3 mol / L (preferably 0.2 mol / L), the pH value is 4.0-5.0 (preferably 4.0), and the volume ratio of the three solutions is 0.8-1.2:0.8-1.2:5-7 (preferably 1:1:6).

[0021] The application of the photo-regulated dual-mimicryse bimetallic carbon-based hybrid obtained by the above preparation method as a peroxide mimicry in the detection of D-alanine (D-Ala) content includes the following steps: First, D-alanine (D-Ala) is mixed with D-amino acid oxidase (DAAO) and reacted for 2-4 h (preferably 3 h). Then, Yb-Gd-CDs, La-Yb-CDs or La-Gd-CDs solutions, 3,3',5,5'-tetramethylbenzidine (TMB) solution, and acetate-sodium acetate buffer are added to the system and mixed well. The mixture is then reacted in a water bath for 2-4 h (preferably 3 h). Due to the different concentrations of H2O2 generated by D-amino acid oxidase catalyzing D-alanine, the hydroxyl radicals (•OH) generated by Yb-Gd-CDs, La-Yb-CDs, or La-Gd-CDs catalyzing H2O2 exhibit different degrees of oxidation color development of TMB, i.e., the color of Ox-TMB is different. The content of D-alanine in the sample is then calculated based on the standard curve of the absorbance value of Ox-TMB at a wavelength of 652 nm and the concentration of D-alanine.

[0022] Further, D-alanine and D-amino acid oxidase are added in a volume ratio of 0.9 to 1.1:1, wherein the concentration of D-alanine is 100 µmol / L to 10 mmol / L, and the concentration of D-amino acid oxidase is 0.04 to 0.06 mg / mL (preferably 0.05 mg / mL), and the water bath reaction temperature is 35 to 40°C (preferably 37°C); Yb-Gd-CDs, La-Yb-CDs, or La-Gd-CDs are also used. The solution concentration is 2-3 mg / mL (preferably 2.44 mg / mL), the concentration of 3,3',5,5'-tetramethylbenzidine (TMB) is 4-6 mmol / L (preferably 5 mmol / L), the concentration of acetate-sodium acetate buffer is 0.1-0.3 mol / L (preferably 0.2 mol / L), the pH value is 4.0-5.0 (preferably 4.0), and the volume ratio of the three solutions is 2-4:4-6:28-35 (preferably 3:5:32).

[0023] The application of the photo-regulated dual-mimicryse bimetallic carbon-based hybrid obtained by the above preparation method as an oxide mimicry in detecting the content of antioxidant small molecules includes the following steps: First, mix Yb-Gd-CDs, La-Yb-CDs or La-Gd-CDs solution, acetate-sodium acetate buffer, and TMB solution, and react for 30-60 min (preferably 40 min). Then, add the same volume of different concentrations of antioxidant small molecules (ascorbic acid, cysteine, and glutathione), mix well, and react for 10-30 min (preferably 20 min). Observe the color change of the solution, and use a 96-well microplate reader to measure the absorbance of the reaction solution at a wavelength of 652 nm. Scan the ultraviolet-visible absorption spectrum in the wavelength range of 500 nm to 800 nm.

[0024] Furthermore, the volume ratio of the Yb-Gd-CDs, La-Yb-CDs or La-Gd-CDs solution, the acetate-sodium acetate buffer solution, and the TMB solution is 4~6:75~80:6~10 (preferably 5:77:8), wherein the concentration of the Yb-Gd-CDs, La-Yb-CDs or La-Gd-CDs solution is 2~3 mg / mL (preferably 2.44 mg / mL), the concentration of the acetate-sodium acetate buffer solution is 0.1~0.3 mol / L (preferably 0.2 mol / L), its pH value is 4.0~5.0 (preferably 4.0), and the concentration of the TMB solution is 4~6 mmol / L (preferably 5 mmol / L). The oxidation colorimetric reaction of TMB is carried out under ultraviolet light irradiation.

[0025] Furthermore, in the step of reducing and decolorizing TMB by adding antioxidant small molecules, the added volume of the antioxidant small molecules was 100 µL, the concentration of ascorbic acid was 40 µmol / L~320 µmol / L, the concentration of cysteine ​​was 40 µmol / L~220 µmol / L, and the concentration of glutathione was 20 µmol / L~480 µmol / L. The relative change in absorbance ΔA in the reaction system with and without the antioxidant analyte was plotted against the analyte concentration. The content of the antioxidant small molecules could then be calculated using a linear equation.

[0026] The application of the photo-regulated dual-mimicryse bimetallic carbon-based hybrid obtained by the above preparation method as an oxide mimicry in detecting the total antioxidant capacity of cell lysates includes the following steps: First, add one of the following solutions: Yb-Gd-CDs, La-Yb-CDs, or La-Gd-CDs, along with acetate-sodium acetate buffer and TMB solution. Mix well and react for 30-50 min (preferably 40 min). Then, add glutathione solution, HeLa cell lysis buffer, HCT116 cell lysis buffer, L929 cell lysis buffer, and A549 cell lysis buffer. React at room temperature for 40-80 min (preferably 60 min). Finally, measure the absorbance at 652 nm using a microplate reader in a 96-well microplate.

[0027] Further, the volume ratio of Yb-Gd-CDs, La-Yb-CDs or La-Gd-CDs solution, acetate-sodium acetate buffer, and TMB solution is 1~3:35~42:3~5 (preferably 2:39:4), wherein the concentration of Yb-Gd-CDs, La-Yb-CDs or La-Gd-CDs solution is 2~3 mg / mL (preferably 2.44 mg / mL), the concentration of acetate-sodium acetate buffer is 0.1~0.3 mol / L (preferably 0.2 mol / L), its pH value is 4.0~5.0 (preferably 4.0), and the concentration of TMB solution is 4~6 mmol / L (preferably 5 mmol / L). The TMB oxidation colorimetric reaction is carried out under ultraviolet light irradiation.

[0028] Furthermore, in the TMB reduction and decolorization step, the added glutathione solutions of different concentrations serve primarily as model molecules for evaluating the total antioxidant capacity of cell lysates. These concentrations range from 20 µmol / L to 160 µmol / L. The concentrations of the four cell lysates—HeLa, HCT116, L929, and A549—are all 100–120 cells / mL (preferably 10⁶ / mL). The changes in absorbance with and without cell lysates are substituted into a standard curve of absorbance change versus glutathione concentration to determine the glutathione equivalents of the four cell lysates at the same cell concentration. These glutathione equivalents are then used to evaluate the total antioxidant capacity of the four cell lysates.

[0029] The application of the photo-regulated dual-mimicry bimetallic carbon-based hybrid obtained by the above preparation method as an oxide mimicry in the total antioxidant capacity of fruits and juices includes the following steps: First, add Yb-Gd-CDs, La-Yb-CDs or La-Gd-CDs solution, acetate-sodium acetate buffer, and TMB solution, mix well, and react for 40-60 min (preferably 50 min). Then, add the same volume of ascorbic acid (AA) solution of different concentrations to the standard curve group, and add the same volume and dilution factor of Wogan mandarin orange, kiwi, grapefruit, or orange juice solution to the sample group. After reacting at room temperature for 40-80 min (preferably 60 min), the absorbance value at a wavelength of 652 nm is measured using a microplate reader.

[0030] Further, the volume ratio of one of the Yb-Gd-CDs, La-Yb-CDs, or La-Gd-CDs solutions, the acetate-sodium acetate buffer, and the TMB solution is 4~6:75~80:6~10 (preferably 5:77:8), wherein the concentration of the Yb-Gd-CDs, La-Yb-CDs, or La-Gd-CDs solution is 2~3 mg / mL (preferably 2.37 mg / mL), the concentration of the acetate-sodium acetate buffer is 0.1~0.3 mol / L (preferably 0.2 mol / L), its pH value is 4.0~5.0 (preferably 4.0), and the concentration of the TMB solution is 4~6 mmol / L (preferably 5 mmol / L). The TMB oxidation colorimetric reaction is carried out under ultraviolet light irradiation.

[0031] Furthermore, in the Ox-TMB reduction and decolorization step, the added ascorbic acid solution serves primarily as a model molecule for evaluating the total antioxidant capacity of fruits. Its concentration ranges from 20 µmol / L to 200 µmol / L. Juices from four fruits—Wogan, kiwi, grapefruit, and orange—were homogenized and centrifuged. The supernatants were diluted 20–40 times (preferably 30 times) before being used as test solutions. The changes in Ox-TMB absorbance with and without fruit and juice solutions were substituted into a standard curve of absorbance changes versus ascorbic acid concentration to determine the ascorbic acid equivalent concentrations of the four fruit juice solutions at the same dilution factor. These ascorbic acid equivalent concentrations were then used to evaluate the total antioxidant capacity of the four fruit juices.

[0032] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0033] (1) The polyvinylpyrrolidone (PVP) used in this invention serves as a stabilizer, dispersant, and carbon source for synthesizing photo-regulated dual-mimicry enzyme bimetallic carbon-based hybrids Yb-Gd-CDs, La-Yb-CDs, or La-Gd-CDs. It is readily soluble in water and various organic solvents such as alcohols and has excellent properties such as good hygroscopicity, film-forming ability, complexing ability, and physiological compatibility. This results in the synthesized bimetallic carbon-based hybrids having good solubility, stability, and dispersibility, which can be used for the detection of complex matrix samples.

[0034] (2) The bimetallic carbon-based hybrids Yb-Gd-CDs, La-Yb-CDs, or La-Gd-CDs synthesized in this invention all possess dual mimicry properties of peroxidase and oxidase. Both of these mimicry properties are photosensitized, and different analytes can be detected by these two mimicry properties without interference through reasonable light regulation. The peroxidase properties of the above-mentioned bimetallic carbon-based hybrid materials can oxidize TMB well in both light and dark conditions, resulting in obvious color development. The oxidase properties show more obvious oxidation color development in light and are weaker in the dark. Based on this, the detection of D-proline and D-alanine using the peroxidase properties of bimetallic carbon-based hybrids is carried out under light-protected conditions. However, the detection of antioxidant small molecule content and total antioxidant capacity of cell lysate using the oxidase properties of bimetallic carbon-based hybrids, as well as the detection of total antioxidant capacity of fruits and juices using the oxidase properties of bimetallic carbon-based hybrids, are carried out under light conditions.

[0035] (3) This invention utilizes a dual-enzyme mimicry detection method based on bimetallic carbon-based hybrids Yb-Gd-CDs, La-Yb-CDs, or La-Gd-CDs to obtain Ox-TMB solutions of different colors. This color change can be controlled according to the concentration of the target analyte and the concentration of generated H2O2. The detection of the target analyte is achieved through changes in the color or absorbance of the Ox-TMB solution. Compared with existing technologies, this invention is simple to operate, has low detection costs, and high sensitivity, enabling rapid and accurate detection of the target analyte. Furthermore, it has significant reference value for the detection of other biomolecules and disease biomarkers, providing new guidance for the future development of enhanced biosensors and biometric systems. Attached Figure Description

[0036] Figure 1 Transmission electron microscopy (TEM) images and particle size distribution diagrams of bimetallic carbon-based hybrids Yb-Gd-CDs.

[0037] Figure 2 The UV-Vis absorption spectrum (a) of the bimetallic carbon-based hybrid Yb-Gd-CDs and the fluorescence spectrum (b) of the bimetallic carbon-based hybrid Yb-Gd-CDs at the optimal excitation and emission wavelengths.

[0038] Figure 3 Transmission electron microscopy (TEM) images and particle size distribution diagrams of the bimetallic carbon-based hybrid La-Yb-CDs.

[0039] Figure 4X-ray photoelectron spectra of the bimetallic carbon-based hybrid La-Yb-CDs (a) and fluorescence spectra of the bimetallic carbon-based hybrid LaYb-CDs at the optimal excitation and emission wavelengths (b).

[0040] Figure 5 To verify the peroxidase-mimicking activity of the bimetallic carbon-based hybrid Yb-Gd-CDs, an oxidation colorimetric diagram (a) is shown. The corresponding UV-Vis absorption spectrum (b) is shown when Yb-Gd-CDs exhibit peroxidase-mimicking activity. The absorbance bar chart (c) shows the Ox-TMB generated by the oxidation of TMB by hydrogen peroxide using Yb-Gd-CDs as a peroxidase-mimicking enzyme at a wavelength of 652 nm.

[0041] Figure 6 To verify the oxidation colorimetric diagram (a) of the bimetallic carbon-based hybrid Yb-Gd-CDs with oxide mimic enzyme activity, the corresponding UV-Vis absorption spectrum (b) when verifying the oxide mimic enzyme activity of Yb-Gd-CDs, and the absorbance bar graph (c) of Ox-TMB generated by the oxidation of TMB by Yb-Gd-CDs as an oxide mimic enzyme at a wavelength of 652 nm under different pH conditions.

[0042] Figure 7 To verify the peroxidase-mimicking activity of the bimetallic carbon-based hybrid La-Yb-CDs, an oxidation colorimetric diagram (a) is shown. The corresponding UV-Vis absorption spectrum (b) is shown when verifying the peroxidase-mimicking activity of La-Yb-CDs. The absorbance bar chart (c) shows the Ox-TMB generated by the oxidation of TMB by hydrogen peroxide catalyzed by La-Yb-CDs as a peroxidase-mimicking enzyme at a wavelength of 652 nm is also shown.

[0043] Figure 8 To verify the oxidation colorimetric diagram (a) of the bimetallic carbon-based hybrid La-Yb-CDs with oxide mimic enzyme activity, the corresponding UV-Vis absorption spectrum (b) when verifying the oxide mimic enzyme activity of La-Yb-CDs, and the absorbance bar graph (c) of Ox-TMB generated by the oxidation of TMB by La-Yb-CDs as an oxide mimic enzyme at a wavelength of 652 nm under different pH conditions.

[0044] Figure 9 The oxidation colorimetric and UV-Vis absorption spectra of D-proline using bimetallic carbon-based hybrid Yb-Gd-CDs as a peroxidase mimic enzyme are shown in (a), the standard curve for D-proline detection by Yb-Gd-CDs is shown in (b), and the results of D-proline detection in serum samples by YbGd-CDs are shown in (c).

[0045] Figure 10 Oxidation colorimetric and UV-Vis absorption spectra of D-alanine detected by bimetallic carbon-based hybrid Yb-Gd-CDs as an oxide mimic enzyme (a) and standard curve of D-alanine detection by Yb-Gd-CDs (b).

[0046] Figure 11 The UV-Vis absorption spectra (ac) and corresponding standard curves (df) of three antioxidant small molecules, glutathione, cysteine ​​and ascorbic acid, were obtained by using the bimetallic carbon-based hybrid Yb-Gd-CDs as an oxidase mimic enzyme.

[0047] Figure 12 The standard curve (a) and detection results (b) for using bimetallic carbon-based hybrid Yb-Gd-CDs as an oxidase mimic to detect the total antioxidant capacity of cell lysates.

[0048] Figure 13 The standard curve (a) and detection results (b) for using bimetallic carbon-based hybrid La-Yb-CDs as an oxidase mimic to detect the total antioxidant capacity of fruits and juices.

[0049] Figure 14 The UV-Vis absorption spectrum (a) of the bimetallic carbon-based hybrid LaGd-CDs and the fluorescence spectrum (b) of the bimetallic carbon-based hybrid LaGd-CDs at the optimal excitation and emission wavelengths are shown.

[0050] Figure 15 The standard curve (a) and detection results (b) of the bimetallic carbon-based hybrid LaGd-CDs as a peroxidase mimic for the detection of serum glucose. Detailed Implementation

[0051] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, but the present invention is not limited thereto.

[0052] Example 1

[0053] Preparation of bimetallic carbon-based hybrid YbGd-CDs: Weigh 40 mg of polyvinylpyrrolidone (PVP) into a 100 mL flat-bottomed flask, add 4.0 mL of ethylene glycol, and disperse and dissolve it thoroughly for 10 min on a magnetic stirrer at 800 rpm. Add 1.0 mL of 20 mmol / L LYbCl3 aqueous solution dropwise to the above PVP ethylene glycol solution with a concentration of 10 mg / mL, and stir magnetically for 10 min. Then add 1.0 mL of 20 mmol / L LGdCl3 aqueous solution dropwise to the above solution and stir magnetically for 10 min. Add 1.5 µL of HNO3 and 7.5 µL of acetic acid to fine-tune the pH of the solution, and stir magnetically for 10 min. Add 200 µL of 100 mmol / L glutathione as a reducing agent. After the reaction solution system is stirred and dispersed evenly at room temperature, place it in an oil bath and stir at 120 °C for 9 h. After cooling, the photo-regulated dual-mimicryse bimetallic carbon-based hybrid YbGd-CDs is obtained.

[0054] Preparation of bimetallic carbon-based hybrids LaYb-CDs: Weigh 40 mg of polyvinylpyrrolidone (PVP) into a 100 mL flat-bottomed flask, add 4.0 mL of ethylene glycol, and disperse and dissolve thoroughly for 10 min at 800 rpm on a magnetic stirrer. Add 1.0 mL of 20 mmol / L LaCl3 aqueous solution to the above PVP ethylene glycol solution with a concentration of 10 mg / mL, and stir magnetically for 10 min. Add 1.0 mL of 20 mmol / L LybCl3 aqueous solution to the above solution, and stir magnetically for 10 min. Add 1.5 µL of HNO3 and 7.5 µL of acetic acid dropwise to fine-tune the pH of the solution, and stir magnetically for 10 min. Add 400 µL of 100 mmol / L glutathione as a reducing agent. After the reaction solution system is evenly dispersed at room temperature, place it in an oil bath and stir at 120 °C for 6 h. After cooling, the photo-regulated dual-mimicryse bimetallic carbon-based hybrid LaYb-CDs is obtained.

[0055] Preparation of bimetallic carbon-based hybrid LaGd-CDs: Weigh 40 mg of polyvinylpyrrolidone (PVP) into a 100 mL flat-bottom flask, add 4.0 mL of ethylene glycol, and disperse and dissolve the PVP in the solution at 800 rpm for 10 min using a magnetic stirrer. Add 1.0 mL of 20 mmol / L LaCl3 aqueous solution to the above 10 mg / mL PVP ethylene glycol solution, and stir magnetically for 10 min. Add 1.0 mL of 20 mmol / L GdCl3 aqueous solution to the above solution, and stir magnetically for 10 min. Add 1.5 µL of HNO3 and 7.5 µL of acetic acid to adjust the pH of the solution, and stir magnetically for 10 min. Add 400 µL of 100 mmol / L glutathione as a reducing agent. After the reaction solution is stirred and dispersed evenly at room temperature, place it in an oil bath and stir at 120 °C for 9 h. After cooling, the photo-regulated dual-mimicryse bimetallic carbon-based hybrid LaGd-CDs is obtained.

[0056] The bimetallic carbon-based hybrids YbGd-CDs, LaYb-CDs, and LaGd-CDs were characterized using various characterization methods. (See [link to relevant documentation]). Figure 1 The transmission electron microscope (TEM) images of YbGd-CDs show that the particles are relatively uniformly dispersed, with an average particle size of 3 nm. (See also...) Figure 2 It is known that the bimetallic carbon-based hybrid YbGd-CDs exhibits distinct blue-green fluorescence under ultraviolet light, with an optimal excitation wavelength of 380 nm and a corresponding optimal emission wavelength of 455 nm. (See also...) Figure 3 As shown in the transmission electron microscope (TEM) images of LaYb-CDs, the particles in the material are relatively uniformly dispersed, with an average particle size of 11 nm. See also... Figure 4 It is known that the bimetallic carbon-based hybrid LaYb-CDs exhibit distinct blue-green fluorescence under ultraviolet light, with an optimal excitation wavelength of 380 nm and a corresponding optimal emission wavelength of 453 nm. (See also...) Figure 5 From a and b, we can see that H2O2 alone cannot oxidize TMB to produce a color change. However, the addition of YbGd-CDs catalyzes the generation of hydroxyl radicals from H2O2, oxidizing TMB to blue Ox-TMB, thus exhibiting its peroxidase-mimicking properties. Figure 5 c indicates that the peroxidase-mimicking activity of YbGd-CDs is strongest at pH 4.0. (See also...) Figure 6 As shown in a and b, YbGd-CDs can oxidize TMB alone to produce a colorimetric result, thus exhibiting oxidative enzyme-like properties. Figure 6 c indicates that the bimetallic carbon-based hybrid Yb-Gd-CDs exhibits the strongest oxidase-mimicking activity at pH 4.0. Figure 7From a and b, we know that the addition of LaYb-CDs catalyzes the generation of hydroxyl radicals from H2O2, oxidizing TMB to blue Ox-TMB, thus exhibiting its peroxidase-mimicking enzyme properties. Figure 7 c indicates that LaYb-CDs exhibit the strongest peroxidase-mimicking catalytic activity at pH 4.0. Figure 8 As shown in a and b, LaYb-CDs can oxidize TMB alone to produce a colorimetric result, thus exhibiting oxidative enzyme-like properties. Figure 8 c indicates that the bimetallic carbon-based hybrid LaYb-CDs exhibits the strongest oxidase-mimicking activity at pH 4.0. (See also...) Figure 14 It is known that the bimetallic carbon-based hybrid LaGd-CDs exhibits obvious blue-green fluorescence under ultraviolet light, with an optimal excitation wavelength of 380 nm and a corresponding optimal emission wavelength of 449 nm.

[0057] Example 2

[0058] Bimetallic carbon-based hybrids YbGd-CDs are used as peroxidase mimics for the detection of D-proline (D-Pro). Add 100 µL of 0.05 mg / mL D-amino acid oxidase (DAAO) to brown centrifuge tubes of different numbers, respectively. Then add 100 µL of D-proline (D-Pro) with initial concentrations of 0 µmol / L, 50 µmol / L, 100 µmol / L, 200 µmol / L, 300 µmol / L, 400 µmol / L, 500 µmol / L, 1 mmol / L, 2 mmol / L, 4 mmol / L, 6 mmol / L, 8 mmol / L, and 10 mmol / L, respectively. Vortex to mix, and incubate at 37 °C for 2.5 h. Then add 100 µL of 2.44 mg / mL LYbGd-CDs solution, 100 µL of 5 mmol / L TMB, and 600 µL of 0.2 mol / L acetate-sodium acetate buffer (pH=4.0), respectively. Vortex to mix, and incubate at 37 °C for another 2.5 h. After the reaction, the color change of the solution was observed. The absorbance value at a wavelength of 652 nm was measured using a 96-well microplate reader, and the UV-Vis absorption spectrum in the wavelength range of 500 nm to 800 nm was scanned. The detection results are as follows: Figure 9 As shown in Figure a, with increasing D-proline concentration, both the colorimetric intensity and UV-Vis absorption intensity of the hydroxyl radicals generated from H₂O₂ catalyzed by the bimetallic carbon-based hybrid YbGd-CDs oxidizing TMB gradually increase. Figure 9b. By plotting a linear standard curve of the generated Ox-TMB absorbance value versus D-proline concentration, the content of D-proline in the unknown sample can be calculated based on the linear equation. The limit of detection (LOD) for D-proline on the linear standard curve is 0.0125 µmol / L.

[0059] Detection of D-proline in serum samples: Add 100 µL of 0.05 mg / mL D-amino acid oxidase (DAAO) to brown centrifuge tubes of different numbers. Then, add 100 µL of each of the seven serum samples (serum-1 to serum-7) diluted 100-fold. Vortex to mix, incubate at 37°C for 2.5 h, then add 100 µL of 2.44 mg / mL LYbGd-CDs solution, 100 µL of 20 mmol / L TMB, and 600 µL of 0.2 mol / L acetate-sodium acetate buffer (pH=4.0). Vortex to mix, and incubate again at 37°C for 2.5 h. After the reaction, observe the color change of the solution and measure the absorbance at 652 nm using a microplate reader. Figure 9 As shown in c, the concentration of D-Pro in the seven serum samples diluted 100 times was calculated using the standard curve of D-Pro, and the concentration of D-Pro in the original serum was obtained after conversion.

[0060] Example 3

[0061] Bimetallic carbon-based hybrid YbGd-CDs are used as peroxidase mimics for the detection of D-alanine (D-Ala). Add 100 µL of 0.05 mg / mL DAAO to brown centrifuge tubes of different numbers, followed by 100 µL of D-alanine (D-Ala) at initial concentrations of 0 µmol / L, 100 µmol / L, 200 µmol / L, 300 µmol / L, 500 µmol / L, 1 mmol / L, 2 mmol / L, 3 mmol / L, 4 mmol / L, 5 mmol / L, 6 mmol / L, 8 mmol / L, and 10 mmol / L, respectively. Vortex to mix, and incubate at 37 °C for 3 h. Then add 60 µL of 2.44 mg / mL LYb-Gd-CDs solution, 100 µL of 5 mmol / L TMB, and 640 µL of 0.2 mol / L acetate-sodium acetate buffer (pH=4.0), respectively. Vortex to mix, and incubate again at 37 °C for 3 h. After the reaction was completed, the color change of the solution was observed, and the absorbance value at a wavelength of 652 nm was measured using a 96-well microplate and an ELISA reader. The UV-Vis absorption spectrum in the wavelength range of 500 nm to 800 nm was scanned.

[0062] Test results as follows Figure 10 As shown in Figure a, with increasing D-alanine concentration, both the colorimetric intensity and UV-Vis absorption intensity of the bimetallic carbon-based hybrid YbGd-CDs catalyzing the oxidation of TMB by hydroxyl radicals from H2O2 gradually increase. Figure 10 b. By plotting a linear standard curve of the generated Ox-TMB absorbance value versus D-alanine concentration, the content of D-alanine in the unknown sample can be calculated based on the linear equation. The limit of detection (LOD) for D-proline on the linear standard curve is 0.0734 µmol / L.

[0063] Example 4

[0064] Bimetallic carbon-based hybrids YbGd-CDs are used as oxidase mimics for detecting antioxidant small molecules. Add 50 µL of 2.44 mg / mL LYb-Gd-CDs solution, 770 µL of NaOAc buffer (0.2 mol / L, pH 4.0), and 80 µL of 5 mmol / L TMB to centrifuge tubes with different numbers. React under UV light for 40 min. Then, add 100 µL of different concentrations of antioxidant small molecules (glutathione, cysteine, and ascorbic acid) to the centrifuge tubes sequentially, vortex, and react for 20 min. Observe the color change of the solution. Measure the absorbance of the reaction solution at 652 nm using a 96-well microplate reader. Scan the UV-Vis absorption spectrum in the wavelength range of 500 nm to 800 nm. Plot a linear standard curve of the relative change ΔA of Ox-TMB absorbance versus analyte concentration with and without antioxidant small molecules. The content of antioxidant small molecules can be calculated based on the linear equation. The detection results are as follows: Figure 11 As shown in Figure ac, with increasing concentrations of the three antioxidant small molecules—glutathione, cysteine, and ascorbic acid—oxidized TMB (Ox-TMB) is gradually reduced and fades, exhibiting a trend of gradually decreasing UV-Vis absorption intensity. Figure 11 According to data from df, the limit of detection (LOD) of the standard curve for detecting glutathione (GSH) is 3.208 µmol / L, the limit of detection (LOD) of the standard curve for cysteine ​​(Cys) is 3.531 µmol / L, and the limit of detection (LOD) of the standard curve for ascorbic acid (AA) is 2.976 µmol / L.

[0065] Example 5

[0066] Bimetallic carbon-based hybrid YbGd-CDs were used as oxidase mimics to detect the total antioxidant capacity (TAC) of cell lysates. Add 40 µL of 2.44 mg / mL LYb-Gd-CDs solution, 780 µL of 0.2 mol / L HAc-NaOAc buffer (pH=4.0), and 80 µL of 5 mmol / L TMB sequentially to centrifuge tubes. Vortex to mix, and irradiate with UV light for 40 min. Then, add 100 µL of glutathione solutions of different concentrations (original concentrations of 20 µmol / L, 40 µmol / L, 60 µmol / L, 80 µmol / L, 100 µmol / L, 120 µmol / L, 140 µmol / L, and 160 µmol / L) sequentially to the standard curve group. Add 100 µL of HeLa, HCT116, L929, and LA549 cell lysis buffers to the cell lysis buffer group. Vortex to mix, and incubate at room temperature for 60 min. Measure the absorbance at 652 nm using a microplate reader in a 96-well microplate. Figure 12 As shown in Figure a, by plotting a linear standard curve of the absorbance change of Ox-TMB versus glutathione concentration, the content of glutathione in the unknown sample can be calculated based on the linear equation. The limit of detection (LOD) for glutathione on the linear standard curve is 1.587 µmol / L.

[0067] The change in Ox-TMB absorbance ∆A with and without cell lysis buffer in the reaction system was substituted into the standard curve 12a for detecting the antioxidant model molecule glutathione to obtain the glutathione equivalent concentration of the four cell lysis buffers at the same cell concentration. The total antioxidant capacity of the four cell lysis buffers was evaluated using the corresponding glutathione equivalent concentration (results are shown in Figure 12a). Figure 12 b).

[0068] Example 6

[0069] Bimetallic carbon-based hybrids LaYb-CDs were used as oxidase mimics to detect the total antioxidant capacity (TAC) of fruits and juices. Add 50 µL of 2.37 mg / mL La-Yb-CDs solution, 770 µL of 0.2 mol / L HAc-NaOAc buffer (pH=4.0), and 80 µL of 5 mmol / L TMB to centrifuge tubes respectively. Vortex and react under UV light for 50 min. For the standard curve group, add 100 µL of ascorbic acid (AA) solution of different concentrations (original concentrations of 20 µmol / L, 40 µmol / L, 60 µmol / L, 80 µmol / L, 100 µmol / L, 120 µmol / L, 140 µmol / L, 160 µmol / L, and 200 µmol / L respectively). For the sample group, add 100 µL of pre-treated tangerine, kiwi, grapefruit, and orange juice solutions diluted 30 times. After reacting at room temperature for 60 min, measure the absorbance at 652 nm using a microplate reader.

[0070] like Figure 13 As shown in Figure a, by plotting a linear standard curve of the absorbance change of Ox-TMB versus the concentration of ascorbic acid, the ascorbic acid content in the unknown sample can be calculated based on the linear equation. The limit of detection (LOD) of the linear standard curve for ascorbic acid is 1.162 µmol / L.

[0071] Substitute the changes in Ox-TMB absorbance in the reaction system with and without fruit and juice solutions into the standard curve of absorbance change versus ascorbic acid concentration. Figure 13 In step a), the ascorbic acid equivalent concentrations (AECs) of the four fruit juice solutions at the same dilution factor were obtained. The total antioxidant capacity of the four fruit juices was evaluated using the corresponding AECs (results are shown in Figure a). Figure 13 b).

[0072] Example 7

[0073] Bimetallic carbon-based hybrid LaGd-CDs are used as peroxidase mimics for serum glucose detection. Add 100 µL of 1 mg / mL glucose oxidase solution to brown centrifuge tubes. For the standard curve group, add 100 µL of glucose solutions of different concentrations (0 µmol / L to 1 mmol / L) sequentially. For the sample group, add 100 µL of each of the four serum sample solutions diluted 8-fold. After mixing, incubate at 37 ℃ for 60 min. Then add 50 µL of 6.24 mmol / L LaGd-CDs solution, 50 µL of 10 mmol / L TMB solution, and 700 µL of NaOAc buffer (0.2 mol / L, pH=4.0), respectively. Mix well and incubate again at 37 ℃ for 60 min. Measure the absorbance at 652 nm using a microplate reader.

[0074] like Figure 15 As shown in Figure a, by plotting a linear standard curve of the absorbance change of Ox-TMB versus glucose concentration, the glucose content in the unknown sample can be calculated based on the linear equation. The limit of detection (LOD) for glucose on the linear standard curve is 9.424 µmol / L.

[0075] Substitute the Ox-TMB absorbance value of the serum sample into the above standard curve for glucose detection. Figure 15 The glucose concentration in the diluted serum sample is obtained in step a), and multiplied by the corresponding dilution factor to obtain the glucose content in the original serum sample (the result is as follows). Figure 15 b).

Claims

1. A method for preparing a photo-regulated dual-mimicry enzyme bimetallic carbon-based hybrid, characterized in that, The light-regulated dual-mimic enzyme bimetallic carbon-based hybrid includes Yb-Gd-CDs, La-Yb-CDs, or La-Gd-CDs, and the preparation method includes the following steps: S1. Dissolve PVP in ethylene glycol and stir until fully dissolved to obtain a PVP ethylene glycol solution; S2. Add two of the following to the PVP glycol solution obtained in S1: ytterbium chloride aqueous solution, lanthanum chloride aqueous solution, and gadolinium chloride aqueous solution. Add the two solutions simultaneously, or add one and disperse it evenly before adding the other. S3. Under stirring conditions, add HNO3 and / or acetic acid to the solution obtained in S2 to adjust the pH of the solution, and then add glutathione solution and stir evenly. The molar ratio of glutathione to bimetallic lanthanum ytterbium, ytterbium gadolinium or lanthanum gadolinium is 1.1~1.3:0.9~1.

1. S4. The solution obtained in S3 is magnetically stirred in an oil bath at 100~140℃. After cooling, pale yellow light-regulated dual-mimicryse bimetallic carbon-based hybrids Yb-Gd-CDs, La-Yb-CDs, or La-Gd-CDs with blue-green fluorescence are obtained.

2. The method for preparing the photo-regulated dual-mimicry enzyme bimetallic carbon-based hybrid according to claim 1, characterized in that, In S1, the ratio of polyvinylpyrrolidone to ethylene glycol is 30~50 mg: 3.0~5.0 mL.

3. The method for preparing the photo-regulated dual-mimicry enzyme bimetallic carbon-based hybrid according to claim 1, characterized in that, In S2, equal amounts of the two solutions are added, and the concentration of the ytterbium chloride aqueous solution, lanthanum chloride aqueous solution, or gadolinium chloride aqueous solution is 10~30 mmol / L.

4. The method for preparing the photo-regulated dual-mimicry bimetallic carbon-based hybrid according to claim 1, characterized in that, In S3, the concentration of glutathione is 80~120 mmol / L.

5. The method for preparing the photo-regulated dual-mimicry enzyme bimetallic carbon-based hybrid according to claim 1, characterized in that, In S4, the reaction time is 5~10h.

6. The application of the photoregulated dual-mimicry bimetallic carbon-based hybrid obtained by any one of claims 1 to 5 as an oxide mimicry in the total antioxidant capacity of fruits and juices, comprising the following steps: First, add Yb-Gd-CDs, La-Yb-CDs or La-Gd-CDs solution, acetate-sodium acetate buffer, and TMB solution, mix well, and react for 40-60 min. Then, add the same volume of ascorbic acid solution of different concentrations to the standard curve group, and add the same volume and dilution factor of Wogan mandarin orange, kiwi, grapefruit, or orange juice solution to the sample group. After reacting at room temperature for 40-80 min, measure the absorbance value at 652 nm using a microplate reader.

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