A fluorescently stained nano-biomass carbon, its staining method and concentration analysis method

Abstract

The present invention discloses a fluorescently stained nano-biomass carbon and its staining method and concentration analysis method, belonging to the technical field of nanomaterials. It includes dissolving 4-chloro-7-nitro-2,1,3-benzoxadiazole (i.e., NBD-Cl) in an organic solvent to obtain a staining solution, and then putting the nano-biomass carbon into the staining solution for staining; the staining method includes: shaking the mixture of the nano-biomass carbon and the staining solution and heating it to T1, maintaining the temperature at T1 for staining for more than 20 minutes, and then placing the mixture in an environment at a temperature of T2 for staining for more than 5 minutes; T1 ≥ 25 °C, T2 ≤ 5 °C. The present invention can perform thermal staining treatment on the nano-biomass carbon sample through the staining solution, with stable staining effect, fast speed, and the staining process only takes about 40 minutes.

Description

Technical Field

[0001] The present invention belongs to the technical field of nanomaterials, and more specifically, relates to a fluorescently stained nano-biomass carbon and its staining method and concentration analysis method. Background Art

[0002] Biochar, also known as bio-black carbon, biomass carbon, and biomass coke, is a class of insoluble, stable, highly aromatic, and carbon-rich solid substances formed by the pyrolysis of biological residues under low-oxygen or oxygen-free environmental conditions. Biochar shows great application potential in soil improvement and environmental pollution remediation. After being applied to the soil, biochar changes the properties of the soil, improves the availability and retention capacity of nutrients in the soil, and thus increases the crop yield. At the same time, in recent years, due to the impact of climate change caused by the emission of greenhouse gases such as carbon dioxide, nitrous oxide, and methane, scientists have begun to pay attention to the use of biochar because it helps to capture and remove greenhouse gases in the atmosphere through the method of biochar sequestration, convert them into a very stable form, and store them in the soil for thousands of years.

[0003] With the development of nanotechnology, people have begun to generate nano-biomass carbon (Nano-BC) for soil and agricultural applications in a sustainable manner. The difference between ordinary biochar and nano-biomass carbon lies in the change of structure with physical and chemical properties. Reducing the particle size of biochar to the micro / nano scale is an effective way to improve its porosity, specific surface area (SSA), fluidity and other properties, and it is also an effective way to improve sub-optimal biochar to the ideal high-quality nanomaterial, namely nano-biomass carbon. The preparation and application of nano-biomass carbon have attracted more and more attention. Therefore, understanding its content and distribution in the nano-biomass carbon environment is of great significance for improving the cost-effectiveness of biochar and understanding and evaluating its ecological health risks.

[0004] Due to the complex chemical composition of nano-biomass carbon and the difficulty in distinguishing nano-biomass carbon from other forms of organic matter, the currently adopted methods are either labor-intensive or require specific detection instruments. These two methods are not practical for most scientific routine analyses. For example, nano-biomass carbon in the soil is manually classified and then analyzed by nuclear magnetic resonance spectroscopy or infrared spectroscopy, or inorganic substances and abiotic carbon are preferentially removed by selective oxidation or acid treatment, and then the remaining organic matter and biochar are analyzed by nuclear magnetic resonance, optical or mass spectrometry. These methods are not suitable for the routine analysis of nano-biomass carbon in the environment by most agronomists or ecologists engaged in environmental research. Therefore, there is an urgent need to establish a simple and rapid method to quantify the concentration of nano-biomass carbon in the environment.

[0005] 4-Chloro-7-nitro-2,1,3-benzoxadiazole (NBD-Cl) is a pale yellow solid that is readily soluble in organic solvents. Because the structure of NBD-Cl contains nitrobenzoxadiazole (NBD), it emits green-yellow fluorescence (λex, maximum = approximately 470 nm, λem, maximum = approximately 530 nm). Recent studies have found that NBD-Cl can stain biochar, so it can be used for the fluorescence staining of nano-biochar. Existing research has carried out the work of staining microplastics with Nile red and observing under a fluorescence microscope, and automatically counting through image recognition software. However, there has been no work on staining nano-biochar, and the existing fluorescence staining methods have deficiencies such as unstable fluorescence staining effect, slow staining speed, and poor resistance to corrosion in acidic and alkaline environments. Summary of the Invention

[0006] 1. Problems to be Solved

[0007] Aiming at the problems in the prior art that there is a lack of a method for staining nano-biochar and the staining effect of the existing staining methods is poor, the present invention provides a method for fluorescently staining nano-biochar and its staining method; by optimizing the staining process under heating and cooling conditions, the deficiencies of the existing staining are overcome, thereby effectively solving the above problems.

[0008] Aiming at the problem that the concentration of existing nano-biochar is difficult to analyze, the present invention provides a method for analyzing the concentration of nano-biochar. By staining the nano-biochar with the staining method of the present invention and then obtaining a standard curve for quantitative analysis, the above problems are effectively solved.

[0009] 2. Technical Solutions

[0010] In order to solve the above problems, the technical solutions adopted by the present invention are as follows:

[0011] A staining method for fluorescently staining nano-biochar of the present invention includes dissolving 4-chloro-7-nitro-2,1,3-benzoxadiazole (i.e., NBD-Cl) in an organic solvent to obtain a staining solution, and then putting the nano-biochar into the staining solution for staining; the staining method includes: oscillating the mixture of nano-biochar and the staining solution and heating it to T1, keeping it at the temperature of T1 for staining for more than 20 min, and then placing the mixture in an environment with a temperature of T2 for staining for more than 5 min; T1 ≥ 25 °C, T2 ≤ 5 °C.

[0012] The oscillation in the present invention is stirring on a magnetic stirrer or ultrasonic oscillation, the stirring rate is 200 rpm to 400 rpm, and the ultrasonic power is 50 KHz to 150 KHz.

[0013] Preferably, the specific steps of the staining are as follows:

[0014] (1) Heating and dyeing: Shake the mixture of nano-biomass carbon and the dyeing solution and heat it to T1 at a rate of 10°C / min to 20°C / min.

[0015] (2) Heat preservation and dyeing: Keep it at a temperature of T1 = 25°C to 75°C for 20 min to 40 min.

[0016] (3) Cooling and dyeing: Place the mixture obtained in step (2) in an ice-water bath at T2 = 0°C to 4°C and cool it for 5 min to 15 min to obtain fluorescently dyed nano-biomass carbon.

[0017] Preferably, repeat steps (1) to (3) to continue dyeing the nano-biomass carbon 1 to 5 times.

[0018] Preferably, the concentration of 4-chloro-7-nitro-2,1,3-benzoxadiazole is 10 mg / L to 100 mg / L.

[0019] Preferably, the organic solvent includes one or more of methanol, n-hexane, and chloroform.

[0020] Preferably, vacuum filter the dyed mixture through an organic filter membrane with a pore size of 0.45 μm, rinse it with deionized water until the filtrate is clear, collect the solid on the filter membrane and dry it to obtain fluorescently dyed nano-biomass carbon.

[0021] Preferably, first disperse the nano-biomass carbon in a solvent to obtain a nano-biomass carbon suspension with a concentration of 5 mg / mL to 15 mg / L, and then mix the suspension with the dyeing solution to obtain the mixture.

[0022] Preferably, the nano-biomass carbon includes one or more of nano-soybean residue biomass carbon, nano-rape seed cake biomass carbon, nano-rice straw biomass carbon, nano-rice husk biomass carbon, nano-corn straw biomass carbon, nano-corn cob biomass carbon, nano-soybean straw biomass carbon, nano-rape straw biomass carbon, nano-peanut straw biomass carbon, or nano-wheat straw biomass carbon.

[0023] A fluorescently dyed nano-biomass carbon of the present invention, which is prepared by the dyeing method described in the present invention.

[0024] A method for analyzing the concentration of nano-biomass carbon of the present invention, which dyes the nano-biomass carbon by the dyeing method described in the present invention to obtain fluorescently dyed nano-biomass carbon, and judges the concentration of the nano-biomass carbon by detecting the fluorescence intensity of the fluorescently dyed nano-biomass carbon.

[0025] Preferably, the specific steps are as follows:

[0026] (1) Use a fluorescence spectrophotometer to excite the fluorescently stained nano - biochar at a wavelength of 488 nm, measure the fluorescence intensity of the fluorescently stained nano - biochar at wavelengths from 515 nm to 525 nm, and plot the standard curve between the mass concentration of the nano - biochar and the fluorescence intensity.

[0027] (2) Use a fluorescence spectrophotometer to measure the fluorescence intensity of the fluorescently stained nano - biochar in the sample to be tested, and calculate the concentration of the nano - biochar according to the standard curve.

[0028] Preferably, the standard curve is formed by fitting the fluorescence intensities at multiple concentrations of the fluorescently stained nano - biochar, and its fitting linear equation is: y = (0.9 - 1.1)×(400701x + 9862.1).

[0029] 3. Beneficial effects

[0030] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0031] (1) For the staining method of a fluorescently stained nano - biochar of the present invention, NBD - Cl is dissolved in an organic solvent to prepare a staining solution, and the nano - biochar sample is subjected to thermal staining treatment with the staining solution. The staining effect is stable, the speed is fast, and the staining process only takes about 40 minutes.

[0032] (2) For the nano - biochar concentration analysis method of the present invention, based on the staining method of the present invention, it can quantitatively analyze the concentration according to the detected fluorescence intensity of the biochar. The concentration analysis method is accurate and is suitable for studying the mass concentration of nano - biochar in the water environment, providing a detection method for researchers to study the environmental behavior and fate of nano - biochar in water. Description of the drawings

[0033] Figure 1 is the fluorescently stained nano - wheat straw biochar under an inverted microscope;

[0034] Figure 2 is the standard curve of the fluorescently stained nano - wheat straw biochar of the present invention;

[0035] Figure 3 is the fluorescence decay curve (1 - 6 days) of the fluorescently stained nano - wheat straw biochar of the present invention. Detailed implementation manners

[0036] The following detailed description of exemplary embodiments of the present invention refers to the accompanying drawings, which form a part of the description. In the drawings, exemplary embodiments in which the present invention can be implemented are shown by way of example, and the features of the present invention are identified by reference numerals. The following more detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but is merely for illustrative purposes and does not limit the description of the features and characteristics of the present invention, to present the best mode of implementing the present invention, and to enable those skilled in the art to implement the present invention. However, it should be understood that various modifications and variations can be made without departing from the scope of the present invention defined by the appended claims. The detailed description and the drawings should be considered illustrative only and not restrictive. If there are any such modifications and variations, they will fall within the scope of the present invention described herein. In addition, the background art is intended to illustrate the current state of research and significance of the present technology and is not intended to limit the present invention or the application fields of the present application and the present invention.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field to which this invention belongs; the terms used in the description of the present invention herein are for the purpose of describing specific embodiments only and are not intended to limit the present invention.

[0038] The object of the present invention is to solve the problems of difficult concentration testing and high cost in the current research on nano-biochar. The method includes the following steps: First, mix a dye and an organic solvent to prepare a staining working solution; Second, mix the staining working solution with nano-biochar, oscillate and stain, filter and dry to obtain fluorescently stained nano-biochar; Third, stain an unknown sample and measure the fluorescence intensity, and use the standard curve between the concentration of the fluorescently stained nano-biochar and the fluorescence intensity to analyze the mass concentration of nano-biochar in the unknown sample. The results of the fluorescence staining and concentration analysis of nano-biochar show that this method can accurately quantify the mass concentration of nano-biochar in a solution. The present invention has the advantages of simple operation, low cost, and fast analysis speed, provides a short-process, fast and convenient nano-biochar quantitative analysis method, and provides a reliable research means for the study of the migration, transformation and fate of nano-biochar in the environment.

[0039] The present invention will be further described below in conjunction with specific embodiments.

[0040] Example 1

[0041] This example provides a staining method for fluorescently stained nano-biochar, which specifically includes the following steps:

[0042] a) Prepare a staining solution by dissolving 10 mg of NBD-Cl dye in 100 mL of an organic solvent (methanol, chromatographic grade, ≥99.9%), with the NBD-Cl concentration being 100 mg / L.

[0043] b) Suspend 0.5 g of the nano-wheat straw biomass carbon solid in 50 mL of a mixed solution of ultrapure water and dimethyl sulfoxide with a volume ratio of 1:1, and mix well to obtain it.

[0044] c) Add 2 mL of the staining working solution to the nano-biomass carbon suspension to obtain a mixture of nano-biomass carbon and the staining solution. Ultrasonically vibrate the mixture at a power of 100 KHz and heat it to 75 °C at a rate of 15 °C / min, and continuously stain at 75 °C for 30 min.

[0045] d) After the mixture solution is heated for a predetermined time, immediately place it in an ice-water bath at 0 - 4 °C and cool for 10 min. Utilize the characteristics of thermal expansion and contraction to fix the dye molecules in the surface structure of the nano-biomass carbon to enhance the staining effect of the nano-biomass carbon.

[0046] e) Vacuum filter the fluorescently stained nano-biomass carbon through an organic filter membrane with a pore size of 0.45 μm, rinse it 3 times with deionized water until the filtrate is clear, collect the solid on the filter membrane and dry it in an oven at 70 °C to obtain the Figure 1 fluorescently stained nano-biomass carbon as shown.

[0047] This example also provides a method for analyzing the concentration of nano-biomass carbon. Based on the above staining method, it specifically includes:

[0048] f) Use a fluorescence spectrophotometer to excite the stained nano-biomass carbon at a wavelength of 488 nm, and measure the fluorescence intensity of the stained nano-biomass carbon at wavelengths from 515 nm to 525 nm. Plot the standard curve between the mass concentration of nano-biomass carbon and the fluorescence intensity. As Figure 2 shown, this standard curve is formed by fitting the fluorescence intensities at multiple concentrations of fluorescently stained nano-biomass carbon, and its fitting linear equation is: y = (0.9 - 1.1)×(400701x + 9862.1). In this example, y = 400701x + 9862.1 can be selected for calculation, where y is the fluorescence intensity (A.U.) and x is the concentration of the fluorescently stained nano-biomass carbon (mg / L).

[0049] g) Use a fluorescence spectrophotometer to measure the fluorescence intensity of the stained nano-biomass carbon in the sample to be measured, and calculate the concentration of nano-biomass carbon according to the standard curve.

[0050] The results show that the correlation coefficient R of the standard curve 2Reached 0.998. For the detection of a nano-biomass charcoal solution with a known concentration of 110.0 mg / L, the mass concentration result of the nano-biomass charcoal detected by this method was 111.5 mg / L, indicating that the quantitative result of this method is accurate and reliable. As Figure 3 shown, within the time period of 1 to 6 days, the fluorescence intensity of the fluorescently stained nano-wheat straw biomass charcoal remained between 82.1% and 89.8% of the initial fluorescence intensity, indicating that the fluorescently stained nano-wheat straw biomass charcoal prepared by this method has strong stability.

[0051] Example 2

[0052] This example provides a staining method for fluorescently stained nano-biomass charcoal. Its specific steps are basically the same as those in Example 1, with the main difference being:

[0053] In this example, after step d) of Example 1, steps c) and d) were repeated for staining once, that is: the mixture was shaken again and heated to 75 °C at °C / min, and continuously stained at 75 °C for 30 min. After the mixture solution was heated for a predetermined time, it was immediately placed in an ice-water bath at 0 - 4 °C and cooled for 10 min.

[0054] Finally, the fluorescently stained nano-biomass charcoal was obtained, and its fluorescence intensity stability within the time period of 1 to 6 days was detected, and a line graph was plotted in Figure 3 .

[0055] Example 3

[0056] This example provides a staining method for fluorescently stained nano-biomass charcoal. Its specific steps are basically the same as those in Example 1, with the main difference being:

[0057] In this example, after step d) of Example 1, steps c) and d) were repeated for staining twice, that is: the mixture was shaken again and heated to 75 °C at °C / min, and continuously stained at 75 °C for 30 min. After the mixture solution was heated for a predetermined time, it was immediately placed in an ice-water bath at 0 - 4 °C and cooled for 10 min; finally, the mixture was shaken again and heated to 75 °C at °C / min, and continuously stained at 75 °C for 30 min. After the mixture solution was heated for a predetermined time, it was immediately placed in an ice-water bath at 0 - 4 °C and cooled for 10 min.

[0058] Finally, the fluorescently stained nano-biomass charcoal was obtained, and its fluorescence intensity stability within the time period of 1 to 6 days was detected, and a line graph was plotted in Figure 3 .

[0059] Combined with Examples 1 - 3, it can be seen that the staining method of the present invention can not only effectively stain nano-biomass charcoal and perform quantitative analysis of the nano-biomass charcoal concentration, but also has excellent staining stability. Refer to Figure 3, the present invention can further improve the dyeing stability by adopting cyclic heating - heat preservation - cooling dyeing. The applicant found that when the fluorescence intensity of the fluorescently dyed nano - wheat straw biomass carbon obtained in the third cycle (Example 3) remained between 94.7% and 98.4% of the initial fluorescence intensity, the stability of the fluorescently dyed nano - biomass carbon reached saturation, and when the number of cycles was further increased, the effect gain was not significant. Therefore, it was determined that 3 cycles were the optimal implementation mode.

[0060] In other embodiments, the nano - biomass carbon used can also be one or more of the following: nano - soybean residue biomass carbon, nano - rapeseed cake biomass carbon, nano - rice straw biomass carbon, nano - rice husk biomass carbon, nano - corn straw biomass carbon, nano - corn cob biomass carbon, nano - soybean straw biomass carbon, nano - rapeseed straw biomass carbon, nano - peanut straw biomass carbon or nano - wheat straw biomass carbon. The present invention will not elaborate on this.

[0061] In the foregoing, the present invention has been described in detail in connection with specific exemplary embodiments. However, it should be understood that various modifications and variations can be made without departing from the scope of the present invention as defined by the appended claims. The detailed description and the drawings should be regarded as illustrative only and not restrictive. If there are any such modifications and variations, then they will all fall within the scope of the present invention described herein. In addition, the background art is intended to illustrate the research and development status and significance of the present technology and is not intended to limit the present invention or the application fields of the present application and the present invention.

[0062] More specifically, although the exemplary embodiments of the present invention have been described herein, the present invention is not limited to these embodiments, but includes any and all embodiments that those skilled in the art can recognize as being modified, omitted, for example, combinations between various embodiments, adaptively changed and / or replaced based on the foregoing detailed description. The limitations in the claims can be broadly interpreted according to the language used in the claims and are not limited to the examples described in the foregoing detailed description or during the implementation of the application. These examples should be considered non - exclusive. Any steps recited in any method or process claim can be executed in any order and are not limited to the order set forth in the claim. Therefore, the scope of the present invention should be determined only by the appended claims and their legal equivalents, rather than by the description and examples given above.

[0063] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the definition in this specification shall prevail. When a mass, concentration, temperature, time, or other value or parameter is expressed as a range, a preferred range, or a series of upper preferred values and lower preferred values, this should be understood to specifically disclose all ranges formed by any pairing of any upper range limit or preferred value with any lower range limit or preferred value, whether or not the ranges are separately disclosed. For example, a range of 1-50 should be understood to include any number, combination of numbers, or sub-ranges selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, as well as all fractional values between the above integers, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. Regarding sub-ranges, specifically consider "nested sub-ranges" extending from any endpoint within the range. For example, nested sub-ranges of the exemplary range 1-50 can include 1-10, 1-20, 1-30, and 1-40 in one direction, or 50-40, 50-30, 50-20, and 50-10 in the other direction.

Claims

1. A staining method for fluorescently stained nano-biomass carbon, characterized in that, It includes dissolving 4-chloro-7-nitro-2,1,3-benzoxadiazole in an organic solvent to obtain a staining solution, and then putting the nano biomass carbon into the staining solution for staining; the staining method includes: shaking the mixture of nano biomass carbon and the staining solution and heating it to T1, keeping it at the temperature of T1 for staining for more than 20 minutes, and then putting the mixture in an environment with a temperature of T2 for staining for more than 5 minutes; T1 is 75 °C, and T2 ≤ 5 °C.

2. The staining method of a fluorescently stained nano-biomass carbon according to claim 1, characterized in that, The specific steps of the staining are as follows: (1) Heating and staining: shaking the mixture of nano biomass carbon and the staining solution and heating it to T1 at a rate of 10 °C / min to 20 °C / min; (2) Incubation staining: incubating at the temperature of T1 = 75 °C for 20 minutes to 40 minutes; (3) Cooling and staining: cooling the mixture obtained in step (2) in an ice-water bath at T2 = 0 °C to 4 °C for 5 minutes to 15 minutes to obtain fluorescently stained nano biomass carbon.

3. The staining method of a fluorescently stained nano-biomass carbon according to claim 2, characterized in that, Repeat steps (1) to (3) to continue staining the nano biomass carbon for 1 to 5 times.

4. The staining method of a fluorescently stained nano-biomass carbon according to claim 1, characterized in that, The concentration of the 4-chloro-7-nitro-2,1,3-benzoxadiazole is 10 mg / L to 100 mg / L; The organic solvent includes one or more of methanol, n-hexane, and chloroform.

5. A staining method for fluorescently stained nano-biomass carbon according to claim 1, characterized in that, Vacuum filter the stained mixture through an organic filter membrane with a pore size of 0.45 μm, rinse it with deionized water until the filtrate is clear, collect the solid on the filter membrane and dry it to obtain fluorescently stained nano biomass carbon.

6. A staining method of a fluorescently stained nano-biomass carbon according to any one of claims 1 to 5, characterized in that, First, disperse the nano biomass carbon in a solvent to obtain a nano biomass carbon suspension with a concentration of 5 mg / mL to 15 mg / L, and then mix the suspension with the staining solution to obtain the mixture; The nano biomass carbon includes one or more of nano soybean residue biomass carbon, nano rapeseed cake biomass carbon, nano rice straw biomass carbon, nano rice husk biomass carbon, nano corn straw biomass carbon, nano corn cob biomass carbon, nano soybean straw biomass carbon, nano rape straw biomass carbon, nano peanut straw biomass carbon, or nano wheat straw biomass carbon.

7. A fluorescently stained nano biomass carbon, characterized in that, The fluorescently stained nano biomass carbon is prepared by the staining method described in any one of claims 1 to 6.

8. A method for analyzing the concentration of nano biochar, characterized in that, Use the staining method described in any one of claims 1 to 6 to stain the nano biomass carbon to obtain fluorescently stained nano biomass carbon, and judge the concentration of the nano biomass carbon by detecting the fluorescence intensity of the fluorescently stained nano biomass carbon.

9. The method for analyzing the concentration of nano-biomass carbon according to claim 8, characterized in that, The specific steps are as follows: (1) Use a fluorescence spectrophotometer to excite the fluorescently stained nano biomass carbon at a wavelength of 488 nm, and measure the fluorescence intensity of the fluorescently stained nano biomass carbon at a wavelength of 515 nm to 525 nm, and draw a standard curve between the mass concentration of the nano biomass carbon and the fluorescence intensity; (2) Use a fluorescence spectrophotometer to measure the fluorescence intensity of the fluorescently stained nano biomass carbon in the sample to be measured, and calculate the concentration of the nano biomass carbon according to the standard curve.

10. A method for analyzing the concentration of nano-biomass carbon according to claim 9, characterized in that, The standard curve is formed by fitting the fluorescence intensities at multiple concentrations of the fluorescently stained nano biomass carbon, and its fitting linear equation is: y = (0.9 to 1.1) × (400701x + 9862.1).

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