Preparation method and application of fluorescent carbon dots based on thermoactinomycetes source
The fluorescent carbon dots were prepared by hydrothermal reaction of high-temperature actinomycetes, which solved the problems of high preparation cost and difficulty in detecting aluminum ions in liquor in the existing technology, and achieved low-cost, simple and environmentally friendly preparation of fluorescent carbon dots and highly specific Al3+ detection.
Patent Information
- Application Number
- CN202510612301.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-13
- Publication Date
- 2025-09-19
AI Technical Summary
The existing preparation methods of fluorescent carbon dots are costly and complex to operate, and cannot be effectively applied to the quantitative detection of aluminum ions in liquor.
Fluorescent carbon dots were synthesized by hydrothermal reaction using thermophilic actinomycetes as raw materials. After centrifugation, membrane filtration, dialysis and vacuum freeze-drying, fluorescent carbon dots with high biocompatibility and good uniformity were prepared for the detection of aluminum ions in liquor.
The low-cost, simple and environmentally friendly preparation of fluorescent carbon dots has been achieved. They have excellent structural and fluorescent properties, can specifically identify Al3+, are not interfered by other heavy metals, and are suitable for fluorescent inks and fluorescent probes.
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Figure CN120664528A_ABST
Abstract
Description
Technical Field
[0001] The present invention can be classified into the field of biochemical applications, and specifically provides a method for preparing fluorescent carbon quantum dots based on thermophilic actinomycetes as the main raw material and its use as Al 3+ Applications in fluorescent probes and fluorescent inks. Background Art
[0002] Fluorescent carbon dots (CDs) are a type of zero-dimensional carbon-based nanomaterials with a size of less than 10 nm. 2 / sp 3 Carbon quantum dots (CQDs) are composed of a hybrid carbon skeleton, with a surface rich in functional groups such as hydroxyl and carboxyl groups. Their unique quantum confinement effect and surface-state luminescence mechanism endow them with excellent optical properties. Furthermore, compared with traditional semiconductor quantum dots, they offer numerous advantages, including water solubility, excitation wavelength dependence, resistance to photobleaching, low toxicity, and excellent biocompatibility. This leads to their growing potential for applications in optical devices, electrical devices, bioimaging, biodrug delivery, environmental monitoring, photocatalysis, and metal ion detection.
[0003] At present, the synthesis methods of fluorescent carbon dots can be mainly divided into two categories: top-down and bottom-up. The top-down method is mainly achieved by cutting large-sized carbon materials through physical or chemical means, including arc discharge method, electrochemical oxidation method, etc. This method often has the problem of high cost and complex operation. Corresponding to it is the bottom-up method, which uses small organic molecules (citric acid, glucose, etc.) as precursors, constructs carbon cores through pyrolysis / condensation, and then completes the preparation of carbon dots. It mainly includes hydrothermal method, microwave-assisted method, template method and solvent thermal synthesis method, but there are also problems such as high post-processing cost and unstable product effect. Therefore, it is still important to continue to explore low-cost, natural and friendly carbon dot preparation raw materials and further develop carbon quantum dots with good biocompatibility and stable optical properties.
[0004] Regardless of the preparation method used, achieving uniform synthesis of carbon dots in size is the key to ensuring the effective application of subsequent carbon dots, but this is still difficult with the current preparation technology. Microorganisms represented by thermoactinomycetes are widely distributed in extreme environments such as high temperature and high heat. They have good characteristics such as easy cultivation, rapid growth and metabolism, and relatively uniform bacterial cell volume. Therefore, they are also a type of potential raw materials for achieving efficient, low-cost, and uniform preparation of fluorescent carbon dots. In this context, the present invention aims to utilize thermoactinomycete bacterial cells as a carbon source to synthesize fluorescent carbon dots with low toxicity, better uniformity, and high biocompatibility using the traditional hydrothermal method, further developing its application potential in fluorescent inks, microbial living cell bioimaging, etc., and has good practical application value. Summary of the Invention
[0005] The technical problem to be solved by the present invention is: to provide a preparation method and application of fluorescent carbon dots based on thermophilic actinomycetes, which solves the technical problem that existing fluorescent carbon dots cannot be used for quantitative detection of aluminum ions in liquor.
[0006] The technical solution of the present invention is: a method for preparing fluorescent carbon dots derived from thermophilic actinomycetes, wherein the thermophilic actinomycetes undergo a hydrothermal reaction to produce fluorescent carbon dots derived from thermophilic actinomycetes.
[0007] The reaction temperature of the hydrothermal reaction is 150-200° C., and the reaction time is 5-10 hours.
[0008] The hydrothermal reaction is followed by the steps of centrifugation, membrane filtration, dialysis, and vacuum freeze drying.
[0009] The centrifugal speed is 9000-11000 r / min, and the centrifugal time is 8-12 min.
[0010] The filter membrane used in the membrane filtration is a 0.10-0.25 μm microporous filter membrane.
[0011] The molecular cutoff of the dialysis bag used for the dialysis is 800-1200Da, and the dialysis time is 5-28h.
[0012] The brown powder obtained after the vacuum freeze-drying step is the thermophilic actinomycete fluorescent carbon dots.
[0013] The application of the thermophilic actinomycete fluorescent carbon dots prepared by the method for preparing fluorescent carbon dots derived from thermophilic actinomycetes in detecting the aluminum ion content in liquor.
[0014] The application of the high-temperature actinomycete fluorescent carbon dots prepared by the method for preparing fluorescent carbon dots derived from high-temperature actinomycetes in the preparation of fluorescent writing materials.
[0015] Fluorescence-enhanced Al 3+ The application method of the ion probe is:
[0016] (1) Diluting the fluorescent carbon dot solution with anhydrous ethanol to prepare a fluorescent probe solution with a final concentration of 0.16-0.2 mg / mL;
[0017] (2) Preparation of standard solution:
[0018] Deionized water was used to prepare aluminum ion solutions with different concentration gradients. The same volume of aluminum ion solutions with different concentration gradients was added to the same volume of fluorescent probe solution to obtain a final aluminum ion concentration of 1.00×10 -6 mol / L-80.0×10 -6Several mol / L standard solutions were mixed evenly and reacted at 15°C for 5 min; the concentration of the fluorescent probe in each standard solution was the same;
[0019] Using a fluorescence spectrophotometer, the fluorescence intensity of the standard solution at 400 nm was measured under the condition of an excitation wavelength of 323 nm, which was recorded as F n , n is the number of standard solutions, n is an integer;
[0020] (3) Preparation of blank solution: Add only deionized water to the fluorescent probe solution to obtain a blank solution. Mix well and react at 15°C for 5 min. Under the excitation wavelength of 323 nm, measure the fluorescence intensity at 400 nm, which is recorded as F0.
[0021] (4) Calculate the relative fluorescence intensity of the standard solution: F n / F0, to obtain the linear relationship between the final concentration of aluminum ions in the standard solution and the relative fluorescence intensity of the standard solution;
[0022] (5) Determination of the test solution: add the test solution to the fluorescent probe solution to obtain a test mixed solution, mix well, react at 15°C for 5 minutes, and measure its fluorescence intensity at 400 nm under an excitation wavelength of 323 nm; according to the linear relationship obtained in step (4), the aluminum ion concentration in the test solution is obtained.
[0023] After adding the aluminum ion solution, the final concentration of the thermoactinomycete fluorescent carbon dots was 0-200 μg / mL.
[0024] Beneficial effects of the present invention:
[0025] First, the present invention uses thermophilic actinomycetes as raw materials and utilizes a one-step hydrothermal reaction to synthesize fluorescent carbon dots. The raw materials are derived from nature and are green and non-toxic. The synthesis method is simple and environmentally friendly, and the pretreatment process is simple and easy.
[0026] Secondly, the fluorescent carbon dots obtained by the present invention have excellent structural and fluorescent advantages. Their rich amino groups and fluorescent characteristics make Al 3+ It has a unique and specific response to the fluorescence enhancement of fluorescent carbon dots, is not interfered by other components such as heavy metals, and can be used as fluorescent ink. BRIEF DESCRIPTION OF THE DRAWINGS
[0027] Figure 1 The morphology and structure characterization of the fluorescent carbon dots of thermophilic actinomycetes, where a is the TEM image of the fluorescent carbon dots of thermophilic actinomycetes; b is the particle size distribution diagram of the fluorescent carbon dots of thermophilic actinomycetes;
[0028] Figure 2Characterization of the surface groups and fluorescence properties of the fluorescent carbon dots of thermophilic actinomycetes, where a is the Fourier transform infrared spectrum of the fluorescent carbon dots of thermophilic actinomycetes; b is the fluorescence excitation and emission spectra of the fluorescent carbon dots of thermophilic actinomycetes;
[0029] Figure 3 The XPS and XRD characterizations of the fluorescent carbon dots of thermoactinomycetes are shown in Figure 1, where a is the XPS graph of the fluorescent carbon dots of thermoactinomycetes; b is the XRD graph of the fluorescent carbon dots of thermoactinomycetes;
[0030] Figure 4 Thermoactinomycetes fluorescent carbon dots to Al 3+ The detection specificity diagram is a graph showing the fluorescence intensity changes of thermoactinomycete fluorescent carbon dots under the same concentration of different metal ions;
[0031] Figure 5 For different concentrations of Al 3+ Fluorescence intensity change diagram of fluorescent carbon dots of thermoactinomycetes;
[0032] Figure 6 Fluorescent ink images of thermophilic actinomycetes fluorescent carbon dots, from a to d, are photos of filter paper with "GZU" and "1234" written on them under sunlight, and photos of filter paper with "GZU" and "1234" written on them under ultraviolet light. DETAILED DESCRIPTION
[0033] Reference Figure 1 The present invention provides a method for preparing fluorescent carbon dots based on thermoactinomycetes, comprising the following steps performed in sequence:
[0034] Step S1. Preparation of thermophilic actinomycetes
[0035] The high-temperature actinomycetes used as raw materials for preparing carbon dots in the present invention were isolated and screened by the present invention team from high-temperature Daqu in the core production area of Chishui River sauce-flavored liquor. The strain name and number are Lacyella sacchari4.012, and the collection number of the strain in the China Center for Type Culture Collection is: CCTCC NO M 20211167.
[0036] First, the thermophilic actinomycetes in ISP2 (Streptomyces medium No. 2) medium are centrifuged to remove the medium, then centrifuged three times with pure water, and then resuspended in pure water to obtain a thermophilic actinomycete solution.
[0037] Step S2. subjecting the thermophilic actinomycete solution to a hydrothermal reaction.
[0038] 15-35 mL of the thermophilic actinomycete solution was added to a polytetrafluoroethylene-lined autoclave, which was then placed in an oven for a hydrothermal reaction at 150-200°C for 5-10 hours. If the reaction temperature is below 150°C or the reaction time is less than 5 hours, carbon dots are difficult to form. If the reaction temperature is above 200°C or the reaction time is longer than 10 hours, the surface groups of the carbon dots will change, resulting in a poor fluorescence effect.
[0039] Step S3. Purification
[0040] After the hydrothermal reaction is completed, the solution is naturally cooled to room temperature, and the obtained brown solution is centrifuged at a speed of 9000-11000 r / min for 8-12 minutes, and then further purified with a 0.10-0.25 μm microporous filter membrane. The purified solution is dialyzed with a dialysis bag with a molecular cutoff of 800-1200 Da for 5-28 hours, and vacuum freeze-dried to obtain a brown powder, which is pure high-temperature actinomycete fluorescent carbon dots, which is stored at 4°C for future use.
[0041] Hereinafter, only certain exemplary embodiments are briefly described. As will be appreciated by those skilled in the art, the described embodiments may be modified in various ways without departing from the spirit or scope of the present invention. Therefore, the drawings and description are to be considered as illustrative in nature and not restrictive.
[0042] Example 1 Preparation of Thermoactinomycete Carbon Dots
[0043] Example 1 of the present invention provides a method for preparing thermoactinomycete fluorescent carbon dots, comprising the following steps performed in sequence:
[0044] Step S1. Preparation of thermophilic actinomycete solution
[0045] First, the thermophilic actinomycetes in ISP2 (Streptomyces medium No. 2) medium are centrifuged to remove the medium, then centrifuged three times with pure water, and then resuspended in pure water to obtain a thermophilic actinomycete solution.
[0046] Step S2. subjecting the thermophilic actinomycete solution to a hydrothermal reaction.
[0047] 15 mL of the high-temperature actinomycete solution was added to a polytetrafluoroethylene-lined autoclave, which was then placed in an oven for a hydrothermal reaction at 150° C. for 5 hours.
[0048] Step S3. Purification
[0049] After the hydrothermal reaction, the mixture was naturally cooled to room temperature. The obtained brown solution was centrifuged at 9000 r / min for 8 minutes and then further purified with a 0.10 μm microporous filter membrane. The purified solution was dialyzed for 5 hours using a dialysis bag with a molecular cutoff of 1200 Da and vacuum freeze-dried. The obtained brown powder was pure high-temperature actinomycete fluorescent carbon dots, which was stored at 4°C for future use.
[0050] Example 2 Characterization of Thermoactinomycete Fluorescent Carbon Dots
[0051] The morphology of the fluorescent carbon dots derived from thermoactinomycetes was characterized by transmission electron microscopy (TEM). Figure 1 a is a TEM image of the fluorescent carbon dots of thermoactinomycetes prepared in Example 1. Figure 1 a It can be seen that the fluorescent carbon dots of thermoactinomycetes are approximately spherical and elliptical granular structures with good dispersion and the particle size distribution shows monodisperse nanoparticles; Figure 1 b It can be seen that the particle size distribution range is 0.55-6.05 nm, and the average particle size is about 2.75 nm, which proves that the fluorescent carbon dots of thermoactinomycetes were successfully synthesized;
[0052] Figure 2 a is the infrared spectrum of the fluorescent carbon dots of thermoactinomycetes. Figure 2 a It can be seen that it is at 3200-3500cm -1 A broad band was observed, proving that there may be a characteristic absorption peak of NH / OH in its skeleton; at 2920-3120 cm -1 A broad band was observed, proving the possible presence of a characteristic absorption peak of CH in its skeleton; at 1640 cm -1 A single peak was observed, proving that there may be a characteristic absorption peak of NH in its skeleton; at 1405 cm -1 A single peak was observed, proving that there may be a characteristic absorption peak of -NO2 in its skeleton; at 1065cm -1 A single peak was observed, proving that there may be a characteristic absorption peak of CN in its skeleton.
[0053] Figure 2 b is the fluorescence excitation and emission spectra of the thermoactinomycete fluorescent carbon dots. As can be seen from the figure, the optimal excitation and emission wavelengths of the thermoactinomycete fluorescent carbon dots are 323 nm and 400 nm, respectively.
[0054] Figure 3(a) The three strong peaks at 283.9, 399.1, and 530.5 eV in the full-scale XPS spectrum are attributed to the characteristic binding energy signals of C1s, N1s, and O1s, respectively. Elemental analysis shows that the thermoactinomycete fluorescent carbon dots are primarily composed of C, N, and O, with content of 69.09% (C), 8.83% (N), and 22.08% (O), respectively.
[0055] Figure 3 b The internal structure and crystal morphology of the thermoactinomycete fluorescent carbon dots were further characterized by XRD, showing a diffraction peak (2θ) at 23.1°, corresponding to the lattice spacing of the carbon (002) crystal plane, which has an amorphous carbon structure.
[0056] Example 3 Application of Thermoactinomycete Fluorescent Carbon Dots as Aluminum Ion Fluorescent Probes and Fluorescent Ink
[0057] Example 3 of the present invention provides an application of thermophilic actinomycete fluorescent carbon dots for detecting aluminum ions in liquor, comprising:
[0058] To prepare Al2O3 fluorescent carbon dots for thermoactinomycetes 3+ The fluorescence intensity of the thermoactinomycete fluorescent carbon dots in Example 1 under different metal ions was tested using a fluorescent probe. Figure 4 As shown in the figure, in aqueous solutions of different metal ions with the same concentration of 800 μM, the fluorescent carbon dots of thermoactinomycetes only react with Al 3+ There is an enhancement effect, and other metal ions have little effect on the fluorescence intensity of the thermophilic actinomycete fluorescent carbon dots, indicating that the thermophilic actinomycete fluorescent carbon dots have a high specificity for recognizing aluminum ions in ethanol solution, and the thermophilic actinomycete fluorescent carbon dots can be used as a fluorescent probe for detecting aluminum ions.
[0059] At room temperature, different concentrations of Al 3+ The concentrations of the solutions were 100μM, 200μM, 300μM, 400μM, 500μM, 600μM and 700μM respectively. 900μL of ethanol-water solution with a volume fraction of 53% ethanol was mixed with 1000μL of thermoactinomycetes fluorescent carbon dots and 500μL of different concentrations of Al 3+ The solutions were mixed uniformly, and the fluorescence spectra of each group of samples were measured at room temperature after a reaction time of 15 min (λex=323 nm, λem=400 nm).
[0060] from Figure 5 It can be seen that with the Al 3+ As the concentration increases, Al 3+ The stronger the fluorescence enhancement effect of the fluorescent carbon dots on thermophilic actinomycetes, the higher the F / F0 and Al 3+There is a linear relationship between the concentrations. Linear fitting is performed on the fluorescence intensity corresponding to the concentration range. The obtained standard curve equation is y=0.00083x+1.073, and the fitting linear correlation coefficient R 2 =0.990, it can be seen that Al 3+ There is a good linear relationship between the concentration and the fluorescence intensity, and Al 3+ The concentration had a significant positive effect on the fluorescence intensity.
[0061] Figure 6 Fluorescent ink images of thermophilic actinomycetes fluorescent carbon dots, from a to d, are photos of filter paper with "GZU" and "1234" written on them under sunlight, and photos of filter paper with "GZU" and "1234" written on them under ultraviolet light.
[0062] The aqueous solution of the high-temperature actinomycete fluorescent carbon dots prepared in Example 1 was dipped into a brush and written on filter paper freely and smoothly. The writing had no color under a fluorescent lamp, but bright blue fluorescence was visible under an ultraviolet lamp. Therefore, the carbon quantum dots provided by the present invention can be used as fluorescent writing materials, or fluorescent ink can be made into fluorescent pens to replace commercial toxic fluorescent pens. Figure 6 From a to d, they are photos of filter paper with "GZU" and "1234" written on it containing an aqueous solution of fluorescent carbon dots containing thermophilic actinomycetes under sunlight, and photos of filter paper with "GZU" and "1234" written on it containing an aqueous solution of fluorescent carbon dots containing thermophilic actinomycetes under ultraviolet light.
[0063] Example 4
[0064] Example 4 of the present invention provides a method for preparing thermoactinomycete fluorescent carbon dots, comprising the following steps performed in sequence:
[0065] Step S1. Preparation of thermophilic actinomycete solution
[0066] First, the thermophilic actinomycetes in ISP2 (Streptomyces medium No. 2) medium are centrifuged to remove the medium, then centrifuged three times with pure water, and then resuspended in pure water to obtain a thermophilic actinomycete solution.
[0067] Step S2. subjecting the thermophilic actinomycete solution to a hydrothermal reaction.
[0068] 30 mL of the high-temperature actinomycete solution was added to a polytetrafluoroethylene-lined autoclave, which was then placed in an oven for a hydrothermal reaction at 200° C. for 20 h.
[0069] Step S3. Purification
[0070] After the hydrothermal reaction, the mixture was naturally cooled to room temperature. The obtained brown solution was centrifuged at 11,000 r / min for 10 min and then further purified using a 0.22 μm microporous filter membrane. The purified solution was dialyzed for 20 h using a dialysis bag with a molecular cutoff of 800 Da and vacuum freeze-dried. The obtained brown powder was pure high-temperature actinomycete fluorescent carbon dots, which was stored at 4°C for future use.
[0071] Example 5
[0072] Example 5 of the present invention provides a method for preparing thermoactinomycete fluorescent carbon dots, comprising the following steps performed in sequence:
[0073] Step S1. Preparation of thermophilic actinomycete solution
[0074] First, the thermophilic actinomycetes in ISP2 (Streptomyces medium No. 2) medium are centrifuged to remove the medium, then centrifuged three times with pure water, and then resuspended in pure water to obtain a thermophilic actinomycete solution.
[0075] Step S2. subjecting the thermophilic actinomycete solution to a hydrothermal reaction.
[0076] 20 mL of the high-temperature actinomycete solution was added to a polytetrafluoroethylene-lined autoclave, which was then placed in an oven for a hydrothermal reaction at 180° C. for 8 h.
[0077] Step S3. Purification
[0078] After the hydrothermal reaction, the mixture was naturally cooled to room temperature. The obtained brown solution was centrifuged at 10,000 r / min for 10 minutes and then further purified using a 0.22 μm microporous filter membrane. The purified solution was dialyzed using a dialysis bag with a molecular cutoff of 1,000 Da for 8 hours and vacuum freeze-dried. The obtained brown powder was pure high-temperature actinomycete fluorescent carbon dots, which was stored at 4°C for future use.
[0079] Example 6
[0080] Example 6 of the present invention provides a method for preparing thermoactinomycete fluorescent carbon dots, comprising the following steps performed in sequence:
[0081] Step S1. Preparation of thermophilic actinomycete solution
[0082] First, the thermophilic actinomycetes in ISP2 (Streptomyces medium No. 2) medium are centrifuged to remove the medium, then centrifuged three times with pure water, and then resuspended in pure water to obtain a thermophilic actinomycete solution.
[0083] Step S2. subjecting the thermophilic actinomycete solution to a hydrothermal reaction.
[0084] 25 mL of the high-temperature actinomycete solution was added to a polytetrafluoroethylene-lined autoclave, which was then placed in an oven for a hydrothermal reaction at 190° C. for 6 h.
[0085] Step S3. Purification
[0086] After the hydrothermal reaction, the mixture was naturally cooled to room temperature. The obtained brown solution was centrifuged at 10,000 r / min for 9 minutes and then further purified using a 0.22 μm microporous filter membrane. The purified solution was dialyzed using a dialysis bag with a molecular cutoff of 1,000 Da for 10 hours and vacuum freeze-dried. The obtained brown powder was pure high-temperature actinomycete fluorescent carbon dots, which was stored at 4°C for future use.
[0087] The above are only preferred embodiments of the present invention. It should be understood that the description of the above embodiments is only used to help understand the method and core ideas of the present invention, and is not used to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, etc. made within the ideas and principles of the present invention should be included in the scope of protection of the present invention.
Claims
1. A method for preparing fluorescent carbon dots derived from thermoactinomycetes, characterized by: After the thermophilic actinomycetes undergo hydrothermal reaction, the bacterial fluorescent carbon dots derived from thermophilic actinomycetes are obtained.
2. The method for preparing fluorescent carbon dots derived from thermoactinomycetes according to claim 1, characterized in that: The reaction temperature of the hydrothermal reaction is 150-200° C., and the reaction time is 5-10 hours.
3. The method for preparing fluorescent carbon dots derived from thermoactinomycetes according to claim 1, characterized in that: The hydrothermal reaction is followed by the steps of centrifugation, membrane filtration, dialysis, and vacuum freeze drying.
4. The method for preparing fluorescent carbon dots derived from thermoactinomycetes according to claim 3, characterized in that: The centrifugal speed is 9000-11000 r / min, and the centrifugal time is 8-12 min.
5. The method for preparing fluorescent carbon dots derived from thermoactinomycetes according to claim 3, characterized in that: The filter membrane used in the membrane filtration is a 0.10-0.25 μm microporous filter membrane.
6. The method for preparing fluorescent carbon dots derived from thermoactinomycetes according to claim 3, characterized in that: The molecular cutoff of the dialysis bag used for the dialysis is 800-1200Da, and the dialysis time is 5-28h.
7. The method for preparing fluorescent carbon dots derived from thermoactinomycetes according to claim 3, characterized in that: The brown powder obtained after the vacuum freeze-drying step is the thermophilic actinomycete fluorescent carbon dots.
8. Use of the thermoactinomycete fluorescent carbon dots prepared by the method for preparing fluorescent carbon dots derived from thermoactinomycetes as claimed in any one of claims 1 to 7 in detecting the aluminum ion content in liquor.
9. Use of the thermoactinomycete-derived fluorescent carbon dots prepared by the method for preparing fluorescent carbon dots derived from thermoactinomycetes as claimed in any one of claims 1 to 7 in preparing fluorescent writing materials.