CN112552906A - Preparation method of nitrogen-doped carbon quantum dots in coffee grounds and method for detecting VB12 through fluorescence - Google Patents
Preparation method of nitrogen-doped carbon quantum dots in coffee grounds and method for detecting VB12 through fluorescence Download PDFInfo
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Abstract
The invention discloses a preparation method of nitrogen-doped carbon quantum dots of coffee grounds and a method for detecting VB12 by fluorescence, wherein the preparation method comprises the following steps: removing impurities from waste coffee grounds, then placing the waste coffee grounds into a crucible to dry at 60 ℃, drying, then placing the waste coffee grounds into a muffle furnace to carbonize at 300 ℃ for 2 hours, cooling to room temperature, taking out the waste coffee grounds, then grinding the carbonized coffee grounds into powder to obtain black charcoal powder, then adding the black charcoal powder into an ethanol solution, mixing, carrying out ultrasonic treatment for 5min, centrifuging, extracting supernate and filtering to obtain a coffee carbon quantum dot solution. The prepared nitrogen-doped carbon quantum dots of the coffee grounds can be used for detecting VB12 by fluorescence. The precursor of the carbon quantum dots is waste coffee grounds, is easy to obtain, has low cost and short analysis time, and can be used for rapid and mass detection. In the fluorescent detection of VB12, the pretreatment is simple, the detection accuracy can be ensured without better separation of VB12, the detection is not influenced by other coexisting vitamins, and the specificity to VB12 is strong.
Description
Technical Field
The invention belongs to the technical field of fluorescent probes, and particularly relates to a preparation method of nitrogen-doped carbon quantum dots of coffee grounds and a method for detecting VB12 by fluorescence.
Background
Vitamin B12(VB12, cobalamin) is a dietary micronutrient ingested from meat or animal products, is an essential water-soluble vitamin containing only metal ions, plays an important role in substance metabolism in the body, causes megaloblastic anemia in the body in the absence of vitamin B, and induces various neurological diseases. Therefore, the VB12 detection method with high selectivity and sensitivity has important value.
The existing VB12 detection method mainly comprises the steps of measuring total VB12 by a high performance liquid chromatography-ultraviolet spectroscopy and microbiological analysis method, and an Atomic Absorption Spectroscopy (AAS), enzyme-linked immunosorbent assay (ELISA), spectrophotometry and the like. However, these detection methods have problems such as high detection limit, long analysis time, high price, and many operators. The optical colorimetry and fluorescence detection have outstanding advantages in the aspects of response time, sensitivity, synthesis process, cost and the like, and carbon quantum dots (CDs) are used as a novel luminescent nano material and have unique photoluminescence, chemical stability and excellent biocompatibility in the detection field. However, the current fluorescent detection system based on carbon quantum dots (CDs) has strict requirements on precursor substances and detection environment, long analysis time and high cost, cannot meet the requirement of rapid and mass detection, and has complex detection flow; the specificity to VB12 is not strong, the pretreatment is complex, and the detection accuracy can be ensured only by better separation of VB 12.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention provides a preparation method of nitrogen-doped carbon quantum dots of coffee grounds and a method for detecting VB12 by fluorescence, and aims to solve the problems in the prior art.
In order to achieve the purpose, the invention adopts the technical scheme that:
a preparation method of nitrogen-doped carbon quantum dots of coffee grounds comprises the following steps:
(1) removing impurities from the waste coffee grounds, then putting the waste coffee grounds into a crucible, drying the waste coffee grounds at 60 ℃, then putting the waste coffee grounds into a muffle furnace to carbonize for 2 hours at 300 ℃, and taking out the waste coffee grounds after cooling to room temperature;
(2) grinding the carbonized coffee grounds into powder to obtain black charcoal powder;
(3) adding 1mg of black charcoal powder into 20mL of ethanol solution, mixing, performing ultrasonic treatment, centrifuging, extracting supernatant, and filtering to obtain a coffee carbon quantum dot solution.
The prepared nitrogen-doped carbon quantum dots of the coffee grounds can be used for the fluorescence detection of VB 12.
The invention further provides a method for detecting VB12 by fluorescence of nitrogen-doped carbon quantum dots in coffee grounds, which comprises the following steps:
(1) preparing a plurality of coffee grounds carbon quantum dot ethanol solutions with the concentration of 0.05 mg/mL;
(2) the excitation condition is 360nm, the slit width EX is 5nm, the EM is 10nm, and the maximum emission peak is 420 nm;
(3) measuring the maximum emission light intensity of each coffee grounds carbon quantum dot ethanol solution in a fluorescence spectrophotometer;
(4) drawing a VB12 standard curve: a series of 10 to 106Respectively adding VB12 solution with ng/mL concentration into 100 times of diluent of 10uL different 0.05mg/mL coffee grounds carbon quantum dot ethanol solution, uniformly stirring, standing for 1min, measuring fluorescence intensity in a fluorescence spectrophotometer, and calculating by using the difference between the fluorescence intensity before and after and VB12 concentration to obtain a standard curve;
(5) adding 10uL of substance to be detected into a new 100-fold diluent of 0.05mg/mL coffee grounds carbon quantum dot ethanol solution, stirring uniformly, and standing for 1 min; measuring the fluorescence intensity in a fluorescence spectrophotometer;
(6) making a difference value before and after the carbon quantum dot solution of the coffee grounds of the substance to be detected, and substituting the difference value into a standard curve to obtain the concentration of the substance to be detected VB 12;
the detection limit of the carbon quantum dot ethanol solution of the coffee grounds is 0.61 ng/mL.
Compared with the defects and shortcomings of the prior art, the invention has the following beneficial effects:
the precursor of the carbon quantum dots is waste coffee grounds, the source is wide, the cost is low, the laboratory horizontal dependence is low due to the simple use of instruments, the analysis time is only 1min, and the rapid and large-scale detection requirements are met. In the fluorescent detection of VB12, the pretreatment is simple, the detection accuracy can be ensured without better separation of VB12, the detection is not influenced by coexistence of VC, B2, B6 and B9, the specificity to VB12 is strong, the sensitivity is high, and the detection limit is low (0.61 ng/mL).
Drawings
FIG. 1 is a fluorescence intensity diagram of carbon quantum dots prepared from bran, straw and coffee grounds respectively as raw materials provided by an embodiment of the invention.
FIG. 2 is a graph showing fluorescence intensity of CCDs prepared at different carbonization temperatures and times according to an embodiment of the present invention.
FIG. 3 is a graph of fluorescence intensity of CCDs made at different sonication times as provided by the examples of the present invention.
FIG. 4 is a graph showing the results of fluorescence spectrophotometry of CCDs according to an embodiment of the present invention.
FIG. 5a is a transmission electron microscope image of CCDs provided by an embodiment of the present invention.
FIG. 5b is an X-ray diffraction pattern of CCDs provided in accordance with an embodiment of the present invention.
FIG. 5c is a Fourier Infrared Electron micrograph of CCDs according to an embodiment of the present invention.
FIG. 5d is Zeta diagram of CCDs provided by the embodiment of the present invention, and d1 and d2 are Zeta diagrams of CCDs and CCDs-VB12, respectively.
FIG. 6 is a block diagram of a computer systemThe CCDs fluorescence quenching and VB12 concentration are 10 ng/mL-106Linear relationship diagram in ng/mL range.
FIG. 7 is a graph showing the response of CCDs to various vitamins, according to an embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
1. Material
A 0.22 μm MCE syringe filter, available from BioSharp (fertilizer combining china), cyanocobalamin (VB12) standard available from supelco (bellefonte) (usa), a real sample (vitamin B12 injection) available from the first human hospital (china) in lanzhou, ethanol available from lyong pharmaceutical industry chemical limited (tianjin, china), ultrapure water available from GLP laboratories of lanzhou university, china, all chemicals and solvents available for direct use.
2. Preparation of nitrogen-doped carbon quantum dots in coffee grounds
(1) Collecting waste coffee grounds as precursor substances;
(2) removing impurities from the waste coffee grounds, and then putting the waste coffee grounds into a crucible to dry at 60 ℃;
(3) drying, carbonizing at 300 deg.C for 2 hr in a muffle furnace (gradually heating), cooling to room temperature, and taking out (gradually cooling);
(4) grinding carbonized coffee grounds into powder to obtain black charcoal powder, adding 1mg of black charcoal powder into ethanol solution (99.7%, 20mL), placing in a conical flask for mixing, carrying out ultrasonic treatment for 5min, centrifuging the product at 8000r/min for 10min, extracting supernatant, filtering by a 0.22 mu m MCE syringe to obtain coffee carbon quantum dot (CCDs) solution, and storing at 4-6 ℃.
3. Optimization of production conditions
(1) The preparation of carbon quantum dots (CDs) is carried out by the method with bran, straw and coffee grounds as raw materials, and the fluorescence intensities of the three CDs are shown in figure 1(a1-a 3). In fig. 1(a1), (a2) (a3) show the fluorescence intensities of three CDs prepared from bran, straw and coffee grounds respectively: a3> a2> a 1.
(2) Fluorescence intensity at different carbonization temperatures and times in the preparation of CCDs
The carbonization temperatures were 200 deg.C, 250 deg.C, 300 deg.C, 450 deg.C, and 550 deg.C, and the carbonization times were 1h and 2h, respectively, and the fluorescence intensity of the obtained CCDs is shown in FIG. 2(b1, b2), and the carbonization temperature of 300 deg.C and carbonization time of 2h are preferred according to the fluorescence intensity.
(3) Fluorescence intensity at different sonication times in the preparation of CCDs
The ultrasonic treatment time is 1min, 3min, 5min, 7min, and 9min, respectively, and the fluorescence intensity of the prepared CCDs is shown in FIG. 3, and the ultrasonic treatment time is preferably 5min according to the fluorescence intensity.
4. Characterization of nitrogen-doped carbon quantum dots in coffee grounds
(1) The fluorescence spectrophotometer showed an optimal excitation of 360nm, with a significant emission peak at 420nm (FIG. 4).
(2) The transmission electron microscope showed a uniform shape and particle size in the range of 1-5nm (shown in FIG. 5 (a)).
(3) X-ray diffraction showed a broad peak at 22.5 ° 2 θ, indicating that the prepared carbon quantum dot structure corresponds to the graphite structure (shown in fig. 5 (b)).
(4) Fourier infrared mirror shows that C-N stretching vibration is 1280.1cm-1The absorption band at (A) indicates the natural doping of N atoms in the CCD and, at the same time, at 3418.6cm-1The strong band at (b) corresponds to the stretching of O-H, 1744.3cm-1The band at (a) is related to the stretching of the C-O bond of the carboxyl group, indicating that such oxygen-containing groups are formed on the surface. 1654cm-1The peak at (a) can be attributed to the skeletal oscillation of the unoxidized carbon domains. Still other characteristic peaks were centered at 2923.3cm-1And 722.2cm-1Due to C-H and CH2Stretching (shown in FIG. 5 (c)).
(5) Zeta shows that when CCDs-VB12 show a distinct negative charge (-5.04V), the solution of CCD is electrically neutral, indicating that VB12 reacts with CCDs to form compounds called CCDs-VB12, in which electron transfer occurs (FIG. 5 (d)1) And FIG. 5 (d)2) Zeta plots for CCDs and CCDs-VB12, respectively).
5. VB12 for fluorescence detection of nitrogen-doped carbon quantum dots in coffee grounds
(1) Preparing a plurality of coffee grounds carbon quantum dot ethanol solutions with the concentration of 0.05 mg/mL;
(2) the method has no requirements on PH and temperature, does not need to adjust pH, ensures that the reaction of the material and a target object is not influenced by the pH, has the excitation condition of 360nm, the slit width EX of 5nm, the EM of 10nm and the maximum emission peak of 420 nm;
(3) measuring the maximum emission light intensity of each coffee grounds carbon quantum dot ethanol solution in a fluorescence spectrophotometer;
(4) drawing a VB12 standard curve: a series of 10 to 106Respectively adding VB12 solution with ng/mL concentration into 100 times of diluent of 10uL different 0.05mg/mL coffee grounds carbon quantum dot ethanol solution, uniformly stirring, standing for 1min, measuring fluorescence intensity in a fluorescence spectrophotometer, and calculating by using the difference between the fluorescence intensity before and after and VB12 concentration to obtain a standard curve;
(5) adding 10uL of substance to be detected into a new 100-fold diluent of 0.05mg/mL coffee grounds carbon quantum dot ethanol solution, stirring uniformly, and standing for 1 min; measuring the fluorescence intensity in a fluorescence spectrophotometer;
(6) making a difference value before and after the carbon quantum dot solution of the coffee grounds of the substance to be detected, and substituting the difference value into a standard curve to obtain the concentration of the substance to be detected VB 12;
(7) the linear range of the detection method is as follows: 10 to 106ng/mL, detection limit of carbon quantum dot ethanol solution of coffee grounds: 0.61 ng/mL.
CCDs fluorescence quenching and VB12 concentration are 10 ng/mL-106The linear relationship in the ng/mL range is shown in FIG. 6.
6. Comparison of VB12 detection by CCDs method and VB12 detection by HPLC method
A medical VB12 injection (1 mL: 0.05mg) was used to verify the effectiveness of the CCDs sensor. In view of the linear range and detection limit, the actual samples were diluted to a range of concentrations (0.05mg/mL, 0.1mg/mL, 0.2mg/mL) and CCDs assay VB12 was prepared under optimal assay conditions. Meanwhile, the sample is also detected by a national standard method (GB/T17819-. The results are shown in Table 1.
As can be seen from Table 1, the method established by the present invention can sensitively detect VB12 in the drug injection, and compared with the HPLC method, the analysis time of the CCDs sensor is only 1min for a single sample, and about 40min is required in the HPLC method. On the other hand, the results detected by the CCDs sensors are closer to the actual concentration in the sample. The reproducibility was studied with Relative Standard Deviation (RSD) of 1.21%, 0.15%, 0.51%. This is considered to be within an acceptable range. In short, the CCDs method established by the invention has good accuracy, wide dynamic range and higher detection efficiency.
7. Response of CCDs to different vitamins
The response of CCDs to VC, VB2, VB6, VB9 and VB12 at concentrations of 1mg/mL is shown in FIG. 7, and it is clear that CCDs are not affected by the coexistence of VC, B2, B6 and B9 during detection and have strong specificity to VB 12.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (6)
1. The preparation method of the nitrogen-doped carbon quantum dots of the coffee grounds is characterized by comprising the following steps of:
removing impurities from waste coffee grounds, then placing the waste coffee grounds into a crucible to dry at 60 ℃, drying, then placing the waste coffee grounds into a muffle furnace to be carbonized, taking out the waste coffee grounds after cooling to room temperature, then grinding the carbonized coffee grounds into powder to obtain black charcoal powder, then adding the black charcoal powder into an ethanol solution, mixing the black charcoal powder and the ethanol solution, carrying out ultrasonic treatment, centrifuging, extracting supernate and filtering to obtain a solution of coffee carbon quantum dots.
2. The method for preparing nitrogen-doped carbon quantum dots according to claim 1, wherein the carbonization temperature is 300 ℃ and the carbonization time is 2 hours.
3. The method for preparing nitrogen-doped carbon quantum dots according to claim 1, wherein the concentration of the black charcoal powder in the ethanol solution is 0.05 mg/mL.
4. The method for preparing nitrogen-doped carbon quantum dots in coffee grounds according to claim 1, wherein the ultrasonic time is 5 min.
5. The use of the nitrogen-doped carbon quantum dots of coffee grounds obtained by the method for preparing the nitrogen-doped carbon quantum dots of coffee grounds as claimed in any one of claims 1 to 4 in VB12 fluorescence detection.
6. The method for detecting VB12 by the nitrogen-doped carbon quantum dot fluorescence of the coffee grounds comprises the following steps:
(1) preparing a plurality of coffee grounds carbon quantum dot ethanol solutions with the concentration of 0.05 mg/mL;
(2) the excitation condition is 360nm, the slit width EX is 5nm, the EM is 10nm, and the maximum emission peak is 420 nm;
(3) measuring the maximum emission light intensity of each coffee grounds carbon quantum dot ethanol solution in a fluorescence spectrophotometer;
(4) drawing a VB12 standard curve: a series of 10 to 106Respectively adding VB12 solution with ng/mL concentration into 100 times of diluent of 10uL different 0.05mg/mL coffee grounds carbon quantum dot solution, uniformly stirring, standing for 1min, measuring fluorescence intensity in a fluorescence spectrophotometer, and calculating by using the difference between the fluorescence intensity before and after and VB12 concentration to obtain a standard curve;
(5) adding 10uL of substance to be detected into 100 times of diluent of a new 0.05mg/mL coffee grounds carbon quantum dot solution, stirring uniformly, and standing for 1 min; measuring the fluorescence intensity in a fluorescence spectrophotometer;
(6) and (4) making a difference value before and after the carbon quantum dot solution of the coffee grounds of the substance to be detected, and substituting the difference value into the standard curve to obtain the concentration of the substance to be detected VB 12.
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| CN114684807A (en) * | 2022-03-15 | 2022-07-01 | 浙江大学医学院附属第四医院 | Room-temperature-driven long-wavelength emission fluorescent carbon dot and preparation method and application thereof |
| CN115125001A (en) * | 2022-08-23 | 2022-09-30 | 济南大学 | Preparation method of green luminescent carbon dots |
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| CN114684807A (en) * | 2022-03-15 | 2022-07-01 | 浙江大学医学院附属第四医院 | Room-temperature-driven long-wavelength emission fluorescent carbon dot and preparation method and application thereof |
| CN114684807B (en) * | 2022-03-15 | 2023-08-04 | 浙江大学医学院附属第四医院 | Long wavelength emission fluorescent carbon dot driven at room temperature and preparation method and application thereof |
| CN115125001A (en) * | 2022-08-23 | 2022-09-30 | 济南大学 | Preparation method of green luminescent carbon dots |
| CN115125001B (en) * | 2022-08-23 | 2023-04-28 | 济南大学 | Preparation method of green luminous carbon dots |
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