CN114229827A - Carbon nanodot, dual-mode probe prepared based on carbon nanodot and application of dual-mode probe - Google Patents

Carbon nanodot, dual-mode probe prepared based on carbon nanodot and application of dual-mode probe Download PDF

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CN114229827A
CN114229827A CN202111244410.7A CN202111244410A CN114229827A CN 114229827 A CN114229827 A CN 114229827A CN 202111244410 A CN202111244410 A CN 202111244410A CN 114229827 A CN114229827 A CN 114229827A
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王素华
王凌霄
余龙
卜一鸣
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Guangdong University of Petrochemical Technology
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Abstract

The invention discloses a carbon nanodot, a dual-mode probe (CND) prepared based on the carbon nanodot and application of the carbon nanodot and the CND. According to the invention, the carbon nanodots are prepared by a simple hydrothermal method, the synthesized carbon nanodots have the advantages of good stability, high sensitivity, good selectivity and the like, the color and the fluorescence of the solution are changed after the mercury ions are added, the color and the fluorescence of the solution can return to the initial state immediately after the glutathione is added, and the mercury ions and the glutathione can be accurately detected in real time. The preparation method of the dual-mode probe comprises the following steps: anhydrous citric acid and urea are dissolved in formamide according to a certain proportion, then hydrothermal reaction is carried out for 10 hours at 180 ℃ to synthesize blue fluorescent carbon nanodots, the carbon nanodots can be quenched by mercury ions and highly chelated by the mercury ions and glutathione, and the mercury ions and the glutathione can be effectively detected through the 'off-on' mechanism.

Description

Carbon nanodot, dual-mode probe prepared based on carbon nanodot and application of dual-mode probe
Technical Field
The invention relates to the technical field of carbon nanodot materials, in particular to a carbon nanodot, a dual-mode probe prepared based on the carbon nanodot and application of the dual-mode probe.
Background
Mercury (Hg)2+) As one of global pollutants, they are widely distributed in rocks, soils, atmosphere, water and organic matter. As society develops, human activities release large amounts of mercury, which leads to mercury pollution in ecosystems. The harm of mercury to the human body mainly involves the central nervous system, the digestive system and the kidneys, and mercury can enter the human body through the skin, the respiratory tract and the digestive tract and accumulate in the living body, and can cause injury and death after being exposed to a high-mercury environment for a long time. There are many conventional quantitative methods for the analysis of mercury, including atomic absorption spectroscopy, cold vapor atomic fluorescence spectroscopy, gas chromatography, and electrochemical methods, but it is very difficult to detect mercury in real time due to the complexity, high cost, and powerful technical expertise of the detection process.
Glutathione (GSH) is a physiological factor with a wide range of functions, is widely distributed in various tissues in the human body, and plays an important role in maintaining the physiological functions of cells. Glutathione helps to maintain normal immune system function and has antioxidant effect. In addition, the glutathione can be used for not only therapeutic drugs, but also basic materials of functional foods, and can be widely applied to the functional foods for resisting aging, enhancing immunity, resisting tumors and the like. Many methods for detecting glutathione have been developed, including capillary electrophoresis, high performance liquid chromatography, raman spectroscopy, and the like. However, these methods have disadvantages such as difficulty in synthesis and low sensitivity. The fluorescence method has the advantages of convenient detection, short response time, high sensitivity, good selectivity and the like. Therefore, the construction of the accurate, sensitive and economic mercury ion and glutathione double-signal fluorescence detection method has important significance.
Carbon Nanodots (CND), a novel nanomaterial, have been the subject of much scientific research since their discovery. The carbon nanodots have the advantages of excellent in vivo and in vitro biocompatibility, photobleaching resistance, easiness in surface functionalization and bioconjugation, colloidal stability, environment-friendly synthesis, low cost and the like, and have the potential in the fields of photocatalysis, analyte detection, biological imaging and the like. Although there are reports related to the detection of mercury ions and glutathione by carbon nanodots, most of the problems are to be solved: (1) most of the probes are single-mode detection probes; (2) the synthesis process is complex and the sensitivity is low; (3) real-time detection is not possible.
The invention patent with the patent application number of CN202110837098.6 discloses a fluorescent carbon nanodot, a preparation method and application thereof in cell nucleus targeted imaging, and the fluorescent carbon nanodot is prepared by taking phenylenediamine and alkyl primary amine hydrochloride as raw materials. The fluorescent carbon nanodots prepared by taking phenylenediamine and alkyl primary amine hydrochloride as raw materials have the property of self-targeting cell nucleus, and the addition of the alkyl primary amine hydrochloride can promote microwave-assisted solid-phase reaction to generate the fluorescent carbon nanodots, so that the problems of time consumption of synthesis steps, consumption of a large amount of organic solvents, complex post-treatment and the like are solved, but the problems of single-mode detection and the like are not solved.
Disclosure of Invention
In order to solve the problems, the invention provides a novel carbon nanodot, a preparation method thereof and a fluorescent and colorimetric reversible dual-mode probe prepared based on the carbon nanodot, which is used for detecting mercury and glutathione. According to the invention, the carbon nanodots are prepared by a simple hydrothermal method, the synthesized carbon nanodots have the advantages of good stability, high sensitivity, good selectivity and the like, the color and the fluorescence of the solution are changed after the mercury ions are added, the color and the fluorescence of the solution can return to the initial state immediately after the glutathione is added, and the mercury ions and the glutathione can be accurately detected in real time.
In order to achieve the purpose, the invention adopts the following technical scheme:
a fluorescent carbon nanodot is prepared by dissolving citric acid and urea in formamide for mixing, and preparing the carbon nanodot by a hydrothermal method, wherein the mass ratio of the citric acid to the urea is 1: 2-4.
Preferably, the ratio of the amount of citric acid to urea is 1:2.
In any of the above embodiments, it is preferable that the ratio of the amounts of the citric acid and urea is 1:3.
In any of the above embodiments, it is preferable that the ratio of the amounts of the citric acid and urea is 1: 4.
In any of the above schemes, the heating temperature in the hydrothermal method is preferably 165-195 ℃ for 9-11 h.
In any of the above schemes, the heating temperature in the hydrothermal method is 165 ℃ and the heating time is 11 h.
In any of the above embodiments, the heating temperature in the hydrothermal process is 180 ℃ and the heating time is 10 hours.
In any of the above embodiments, the heating temperature in the hydrothermal process is 195 ℃ and the heating time is 9 h.
The invention also discloses a preparation method of the fluorescent carbon nanodot, which comprises the following steps:
(1) fully mixing urea and anhydrous citric acid, taking formamide as a solvent, then adding the mixture into a reaction kettle, and carrying out hydrothermal reaction to obtain a purple black solution;
(2) and (2) cooling, centrifuging, filtering and dialyzing the solution obtained in the step (1) to obtain the carbon nanodot solution.
Preferably, the ratio of the amount of citric acid to urea in step (1) is 1: 2-4.
In any of the above embodiments, it is preferable that the ratio of the amounts of the citric acid and urea in the step (1) is 1:2.
In any of the above embodiments, it is preferable that the ratio of the amounts of the citric acid and urea in the step (1) is 1:3.
In any of the above embodiments, it is preferable that the ratio of the amounts of the citric acid and urea in the step (1) is 1: 4.
In any of the above schemes, preferably, the ultrasonic treatment is performed for 12-18min before the hydrothermal reaction in the step (1), the solution after the ultrasonic treatment is completely carried out is transferred to a reaction kettle with a polytetrafluoroethylene lining, and the solution is heated at 165-195 ℃ for 9-11 h.
In any of the above schemes, preferably, the ultrasonic time is 12min, the solution after complete ultrasonic treatment is transferred to a 50ml reaction kettle with a polytetrafluoroethylene lining, and heated at 165 ℃ for 11 h.
In any of the above schemes, preferably, the ultrasonic time is 15min, and the solution after complete ultrasonic treatment is transferred to a 50ml reaction kettle with a polytetrafluoroethylene lining and heated at 180 ℃ for 10 h.
In any of the above schemes, preferably, the ultrasonic time is 18min, and the solution after complete ultrasonic treatment is transferred to a 50ml reaction kettle with a polytetrafluoroethylene lining and heated at 195 ℃ for 9 h.
In any of the above schemes, it is preferable that the centrifugation in step (2) is carried out at 8000-12000rpm for 9-11 min.
In any of the above embodiments, it is preferable that the centrifugation in the step (2) is performed at 8000 for 11 min.
In any of the above schemes, preferably, the centrifugation in step (2) is performed at 10000rpm for 10 min.
In any of the above embodiments, it is preferable that the centrifugation in the step (2) is performed at 12000rpm for 9 min.
In any of the above schemes, preferably, in the specific preparation, 0.38g (2mmol) of anhydrous citric acid and 0.36g (6mmol) of urea are dissolved in 20ml of formamide, then ultrasonic treatment is carried out for 15min, the solution after the ultrasonic treatment is completely transferred to a 50ml reaction kettle with a polytetrafluoroethylene lining, heating is carried out at 180 ℃ for 10h, and after the complete reaction, cooling is carried out to room temperature, thus obtaining a purple black solution. To remove most of the solids, the cooled solution was centrifuged at 10000rpm for 10 min. The centrifuged solution was filtered through a 0.22 μ M filter and then dialyzed against a cellulose ester dialysis bag (1000Da) for 24h to remove small molecules and unreacted reagents. The prepared carbon dot solution was then stored hermetically in a refrigerator at 4 ℃.
The invention also discloses a novel dual-mode probe prepared from the fluorescent carbon nanodots, and the preparation method comprises the following steps:
(1) fully mixing urea and anhydrous citric acid, taking formamide as a solvent, then adding the mixture into a reaction kettle, and carrying out hydrothermal reaction to obtain a purple black solution;
(2) cooling, centrifuging, filtering and dialyzing the solution obtained in the step (1) to obtain a carbon nanodot solution;
(3) and mixing the carbon nanodot solution with a phosphate buffer solution to prepare a probe for detecting metal ions, or mixing the carbon nanodot solution with the phosphate buffer solution and then adding a mercury ion standard solution to prepare the probe for detecting amino acids.
Preferably, the ratio of the amount of citric acid to urea in step (1) is 1: 2-4.
In any of the above embodiments, it is preferable that the ratio of the amounts of the citric acid and urea in the step (1) is 1:2.
In any of the above embodiments, it is preferable that the ratio of the amounts of the citric acid and urea in the step (1) is 1:3.
In any of the above embodiments, it is preferable that the ratio of the amounts of the citric acid and urea in the step (1) is 1: 4. In any of the above schemes, preferably, the ultrasonic treatment is performed for 12-18min before the hydrothermal reaction in the step (1), the solution after the ultrasonic treatment is completely carried out is transferred to a reaction kettle with a polytetrafluoroethylene lining, and the solution is heated at 165-195 ℃ for 9-11 h.
In any of the above schemes, preferably, the ultrasonic time is 12min, the solution after complete ultrasonic treatment is transferred to a 50ml reaction kettle with a polytetrafluoroethylene lining, and heated at 165 ℃ for 11 h.
In any of the above schemes, preferably, the ultrasonic time is 15min, and the solution after complete ultrasonic treatment is transferred to a 50ml reaction kettle with a polytetrafluoroethylene lining and heated at 180 ℃ for 10 h.
In any of the above schemes, preferably, the ultrasonic time is 18min, and the solution after complete ultrasonic treatment is transferred to a 50ml reaction kettle with a polytetrafluoroethylene lining and heated at 195 ℃ for 9 h.
In any of the above schemes, it is preferable that the centrifugation in step (2) is carried out at 8000- & ltSUB & gt 12000rpm for 9-11min, the centrifuged solution is filtered through a 0.2-0.24 μ M filter membrane, and then dialyzed with a cellulose ester dialysis bag for 23-25h to remove small molecules and unreacted reagents, and then the prepared carbon dot solution is hermetically stored in a refrigerator at 4 ℃.
In any of the above embodiments, the centrifugation in step (2) is preferably carried out at 8000rpm for 11 min.
In any of the above schemes, preferably, the centrifugation in step (2) is performed at 10000rpm for 10 min.
In any of the above embodiments, it is preferable that the centrifugation in the step (2) is performed at 12000rpm for 9 min.
In any of the above embodiments, the solution after centrifugation is preferably filtered through a 0.2 μ M filter and then dialyzed against a cellulose ester dialysis bag for 23 hours.
In any of the above embodiments, the centrifuged solution is preferably filtered through a 0.22 μ M filter and then dialyzed against a cellulose ester dialysis bag for 24 hours.
In any of the above embodiments, the centrifuged solution is preferably filtered through a 0.24 μ M filter and then dialyzed against a cellulose ester dialysis bag for 25 hours.
In any of the above schemes, preferably, the preparation method of the probe for detecting metal ions in step (3) is: 8-12 mul of carbon dot stock solution and 1.5-2.5ml of phosphate buffer solution are fully mixed and added into a cuvette to prepare a probe solution. The pH of the phosphate buffer solution is 8, so as to remove the interference of part of the metal cation standard solution containing nitric acid.
In any of the above schemes, preferably, the preparation method of the probe for detecting metal ions in step (3) is: mu.l of the stock solution of the carbon spots and 1.5ml of phosphate buffer solution were mixed well and added to the cuvette to prepare a probe solution.
In any of the above schemes, preferably, the preparation method of the probe for detecting metal ions in step (3) is: mu.l of the stock solution with carbon dots and 2ml of phosphate buffer solution are fully mixed and added into a cuvette to prepare a probe solution.
In any of the above schemes, preferably, the preparation method of the probe for detecting metal ions in step (3) is: mu.l of the carbopoint stock solution and 2.5ml of phosphate buffer solution were mixed well and added to the cuvette to prepare a probe solution.
In any of the above embodiments, preferably, the method for preparing the probe for detecting amino acid in step (3) comprises: and (3) fully mixing the carbon dot stock solution and a phosphate buffer solution, adding the mixture into a cuvette, adding 10 mu M of mercury ion standard solution, and finally preparing a probe solution for detecting amino acid.
In any of the above embodiments, preferably, in the method for preparing an amino acid probe for detection in step (3): the consumption of the carbon dot stock solution is 8-12 mu l, the consumption of the phosphate buffer solution is 1.5-2.5ml, and the consumption of the mercury ion standard solution is 8-12 mu M.
In any of the above embodiments, preferably, in the method for preparing an amino acid probe for detection in step (3): the dosage of the carbon dot stock solution is 8 mu l, the dosage of the phosphate buffer solution is 1.5ml, and the dosage of the mercury ion standard solution is 8 mu M.
In any of the above embodiments, preferably, in the method for preparing an amino acid probe for detection in step (3): the dosage of the carbon dot stock solution is 10 mu l, the dosage of the phosphate buffer solution is 2ml, and the dosage of the mercury ion standard solution is 10 mu M.
In any of the above embodiments, preferably, in the method for preparing an amino acid probe for detection in step (3): the dosage of the carbon dot stock solution is 12 mu l, the dosage of the phosphate buffer solution is 2.5ml, and the dosage of the mercury ion standard solution is 12 mu M.
In any of the above schemes, preferably, the metal ion detection bimodal probe prepared in step (3) is used for fluorescence detection and colorimetric detection of mercury ions, and the amino acid detection bimodal probe is used for fluorescence detection and colorimetric detection of glutathione.
The application also provides the application of the dual-mode probe prepared by the preparation method in the aspects of metal cation selection, fluorescence detection and colorimetric detection, and amino acid selection, fluorescence detection and colorimetric detection. Drawing a standard curve for detecting mercury ions, selectively detecting metal cations, drawing a standard curve for detecting glutathione, and selectively detecting amino acids.
The metal cations comprise at least mercury ions and the amino acids comprise at least glutathione.
Preferably, the drawing method of the standard curve for detecting mercury ions comprises the following steps: and taking the metal ion detection dual-mode probe solution, sequentially adding a mercury ion standard solution into the probe solution, respectively recording the changes of fluorescence intensity and absorbance, and drawing a standard change curve.
In any of the above embodiments, the method for selectively detecting metal cations preferably comprises: and under the same condition, taking the metal ion detection dual-mode probe solution, adding a metal ion standard solution into the probe solution, and respectively recording the changes of fluorescence intensity and absorbance. In the metal selectivity detection, glutathione is added to the mixed solution of copper ions and carbon nanodots to eliminate the interference of the copper ions.
In any of the above schemes, preferably, the method for drawing the standard curve for detecting glutathione is as follows: and taking a dual-mode probe solution for detecting amino acid, sequentially adding a glutathione standard solution into the probe solution, respectively recording the changes of fluorescence intensity and absorbance, and drawing a standard change curve.
In any of the above embodiments, preferably, the method for selectively detecting an amino acid comprises: and under the same condition, taking the amino acid detection dual-mode probe solution, adding an amino acid standard solution into the probe solution, and respectively recording the changes of fluorescence intensity and absorbance.
In any of the above embodiments, preferably, the method for detecting sensitivity of mercury ions comprises: (a) and (3) taking 10 mu l of carbon nano-dot solution, diluting the carbon nano-dot solution into a cuvette by using a buffer solution to 2ml, gradually adding a mercury ion standard solution, and respectively obtaining a standard curve of fluorescence intensity and ultraviolet absorption intensity with mercury ion concentration through testing to calculate the sensitivity of the probe for detecting mercury ions.
The pH of the buffer solution is 8, so as to remove the interference of part of the metal cation standard solution containing nitric acid.
In any of the above embodiments, the method for detecting glutathione with sensitivity preferably comprises: and (b) continuously and gradually adding a glutathione standard solution by adopting the mixed solution of mercury ions and carbon nanodots obtained in the step (a), and respectively obtaining a standard curve of fluorescence intensity, ultraviolet absorption intensity and glutathione concentration through testing to calculate the sensitivity of the probe to glutathione detection.
The dual-mode probe prepared by the preparation method is used for detecting mercury ions and glutathione in different water samples. Real water samples including lake water and tap water are detected to evaluate the applicability of the dual-mode probe in the environment.
Compared with the prior art, the invention has the beneficial effects that:
(1) the principle of the invention for detecting mercury ions by fluorescence colorimetry is based on a probe 'closing' strategy, and particularly shows that the probe quenches fluorescence and changes the color of a solution due to the chelation of the mercury ions and the synthesized carbon nanodots. Detection of glutathione a probe-based "turn-on" strategy, due to the strong affinity of the mercuric ions and the thiol bonds in glutathione, the detachment of the mercuric ions, allowing the probe fluorescence and solution color to gradually return to the original state. Therefore, high accuracy and real-time visual detection of mercury ions and glutathione are realized.
The invention provides a method for preparing a dual-mode probe, which comprises the following steps: the method comprises the steps of dissolving anhydrous citric acid and urea in formamide according to a certain proportion, synthesizing a blue fluorescent carbon nano-dot through a hydrothermal reaction, and effectively detecting mercury ions and glutathione through an 'off-on' mechanism by utilizing the carbon nano-dot which can be quenched by mercury ions and the high chelation between the mercury ions and the glutathione.
(2) The carbon nanodot is prepared based on a citric acid, urea and formamide system, the probe with two modes is developed, and the preparation method is simple, low in cost, convenient to operate, high in accuracy and sensitivity and wide in application prospect.
(3) The invention realizes 'one-needle multi-detection', namely mercury ions and glutathione can be well detected, the detection limits of a fluorescence mode are respectively 6.8nM and 10.9nM, and the detection limits of a colorimetric mode are respectively 0.38 mu M and 0.56 mu M.
(4) The carbon nanodots prepared by the method have the advantages of high sensitivity, good selectivity, strong anti-interference capability and low detection limit on mercury ions and glutathione, are used for real-time detection in real water samples, and have practical application value.
Drawings
FIG. 1 is a transmission electron microscope image of carbon nanodots prepared according to the present invention;
FIG. 2 is a diagram of the UV-VIS absorption spectrum and the fluorescence spectrum of the carbon nanodots prepared according to the present invention;
FIG. 3 is an X-ray energy spectrum of carbon nanodots prepared according to the present invention;
FIG. 4 is a graph showing the linear change of the fluorescence response of a probe for detecting metal ions, which is prepared by mixing carbon nanoparticles and a phosphate buffer solution and prepared according to the present invention, to mercury ions at room temperature;
FIG. 5 is a diagram showing the selective fluorescence detection of metal ions by a probe for detecting metal ions prepared by mixing a carbon nanodot solution prepared according to the present invention with a phosphate buffer solution;
FIG. 6 is a graph showing the linear change of fluorescence response of a probe prepared by mixing carbon nanoparticles prepared according to the present invention with a phosphate buffer solution and then adding 10. mu.M of a mercury ion standard solution to detect amino acids to glutathione;
FIG. 7 is a diagram showing selective fluorescence detection of amino acids by a probe prepared by mixing carbon nano-particles prepared by the present invention with a phosphate buffer solution and then adding 10. mu.M of a mercury ion standard solution to detect amino acids;
FIG. 8 is a linear change curve diagram of colorimetric response of a probe for detecting metal ions, prepared by mixing a carbon nanodot solution prepared according to the present invention with a phosphate buffer solution, to mercury ions at room temperature;
FIG. 9 is a diagram showing the selective colorimetric detection of metal ions by a probe for detecting metal ions prepared by mixing a carbon nanodot solution prepared according to the present invention with a phosphate buffer solution;
FIG. 10 is a linear change curve diagram of colorimetric response of a probe prepared by mixing a carbon nano-dot prepared by the present invention with a phosphate buffer solution and then adding 10 μ M of a mercury ion standard solution to detect amino acids to glutathione;
FIG. 11 is a selective colorimetric detection diagram of amino acids with probes for detecting amino acids, prepared by mixing carbon nano-dots prepared by the present invention with a phosphate buffer solution and then adding 10 μ M mercury ion standard solution;
FIG. 12 is a graph showing the change in color of a probe for detecting metal ions, which is prepared by mixing carbon nanoparticles prepared according to the present invention with a phosphate buffer, in real time with the addition of mercury ions;
FIG. 13 is a graph showing the change in color of a probe solution prepared by mixing carbon nanoparticles prepared according to the present invention with a phosphate buffer solution and then adding a 10. mu.M mercury ion standard solution to detect amino acids in real time with the addition of glutathione;
FIG. 14 is a comparison graph of the interfering fluorescence response linear variation curve of the probe for detecting metal ions prepared by mixing the carbon nano-particles prepared by the present invention with a phosphate buffer solution to exclude copper ions.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments.
Example one
1. Preparation of carbon nanodot solution
Dissolving 0.38g (2mmol) of anhydrous citric acid and 0.36g (6mmol) of urea in 20ml of formamide, then carrying out ultrasonic treatment for 15min, transferring the solution after the ultrasonic treatment to a 50ml reaction kettle with a polytetrafluoroethylene lining, heating at 180 ℃ for 10h, carrying out complete reaction, and cooling to room temperature to obtain a purple black solution. To remove most of the solids, the cooled solution was centrifuged at 10000rpm for 10 min. The centrifuged solution was filtered through a 0.22 μ M filter and then dialyzed against a cellulose ester dialysis bag (1000Da) for 24h to remove small molecules and unreacted reagents. The prepared carbon dot solution was then stored hermetically in a refrigerator at 4 ℃.
2. Preparation of fluorescent probe for detecting metal ions
Mu.l of the stock solution was thoroughly mixed with 2ml of phosphate buffer (pH 8) and added to a cuvette to prepare a probe solution.
3. Fluorescent response of probe to mercury ions
In order to illustrate the function of the probe in the mercury ion fluorescence detection, 0-10 mu M of mercury ion standard solution is sequentially added into the probe solution, and the change of the fluorescence intensity is recorded. The fluorescence intensity gradually decreases with increasing concentration of mercury ions.
FIG. 1 is a transmission electron microscope image of a bimodal probe prepared according to the present invention. FIG. 2 is a diagram of the UV-visible absorption spectrum and the fluorescence spectrum of the bimodal probe prepared according to the present invention. FIG. 3 is an X-ray energy spectrum of a bimodal probe prepared according to the present invention.
4. Selectivity of fluorescent probes to metal cations
In order to evaluate the selectivity of the probe for fluorescence detection of mercury ions, Co is sequentially subjected to the detection under the same conditions2+、Cr3+、Cd2+、Cr6+、Al3+、K+、Na+、Ca2+、Pb2+、Zn2+、Mn2+、Cu2+、Ni2+、Mg2+、Fe3+Fluorescence selectivity experiments are carried out on a series of metal cations, and the results show that the probe has good selectivity on mercury ions. FIG. 4 is a graph showing the linear change of the fluorescence response of a probe for detecting metal ions, which is prepared by mixing carbon nanoparticles and a phosphate buffer solution and prepared according to the present invention, to mercury ions at room temperature; FIG. 5 is a diagram showing the selective fluorescence detection of metal ions by the probe for detecting metal ions prepared by mixing the carbon nanodot solution prepared by the present invention with a phosphate buffer solution.
Example two
1. Preparation of carbon nanodot solution
The preparation process of this step is the same as in example one.
2. Preparation of fluorescent probe for detecting amino acid
And (3) fully mixing 10 mu l of carbon dot stock solution with 2ml of phosphate buffer solution (pH 8), adding into a cuvette, adding 10 mu M of mercury ion standard solution, and finally preparing into a probe solution for detecting amino acid.
3. Fluorescent response of probes to glutathione
To illustrate the role of the probe in glutathione fluorescence detection, 0-10. mu.M glutathione standard solution was added to the probe solution in sequence and the change in fluorescence intensity was recorded. The fluorescence intensity gradually increased with increasing glutathione concentration. FIG. 6 is a graph showing the linear change of fluorescence response of a probe prepared by mixing a carbon nano-dot prepared by the present invention with a phosphate buffer solution and then adding a 10. mu.M mercury ion standard solution to detect amino acids to glutathione.
4. Selectivity of fluorescent probes for amino acids
In order to evaluate the selectivity of the probe for detecting glutathione by fluorescence, a series of amino acids including cysteine (Cys), phenylalanine (Phe), glycine (Gly), arginine (Arg), proline (Pro), tryptophan (Trp), serine (Ser), threonine (Thr) and histidine (His) are subjected to fluorescence selectivity experiments in sequence under the same conditions, and the result shows that the probe has good selectivity for biological thiols (GSH and Cys), and although the cysteine recovers the fluorescence, the carbon dot serves as a good probe for detecting glutathione in a dual mode for two reasons. One is that the concentration of glutathione in the biological sample is much higher than cysteine. On the other hand, when the glutathione concentration reached the μ M level, cysteine had reached an ultra-low concentration. FIG. 7 is a selective fluorescence detection diagram of Glutathione (GSH), cysteine (Cys), phenylalanine (Phe), glycine (Gly), arginine (Arg), proline (Pro), tryptophan (Trp), serine (Ser), threonine (Thr), and histidine (His) in a probe prepared by mixing the carbon nano-dots prepared by the present invention with a phosphate buffer solution and then adding 10 μ M of mercury ion standard solution.
EXAMPLE III
1. Preparation of carbon nanodot solution
The preparation process of this step is the same as in example one.
2. Preparation of colorimetric probe for detecting metal ions
The preparation process of this step is the same as in example one.
3. Colorimetric response of probes to mercury ions
In order to illustrate the function of the probe in the mercury ion fluorescence detection, 0-20 mu M of mercury ion standard solution is sequentially added into the probe solution, and the change of absorbance is recorded. The absorbance gradually decreases as the concentration of mercury ions increases. Fig. 8 is a linear change curve diagram of colorimetric response of a probe for detecting metal ions, prepared by mixing the carbon nanodot solution prepared by the present invention and a phosphate buffer solution, to mercury ions at room temperature.
4. Selectivity of colorimetric probes for metal cations
In order to evaluate the selectivity of the probe for colorimetric detection of mercury ions, Co is sequentially subjected to colorimetric detection under the same conditions2+、Cr3+、Cd2+、Cr6+、Al3+、K+、Na+、Ca2+、Pb2+、Zn2+、Mn2+、Cu2+、Ni2+、Mg2+、Fe3+A series of metal cationsColorimetric selectivity experiments are carried out, and results show that the probe has good selectivity on mercury ions. Fig. 9 is a selective colorimetric detection diagram of metal ions by a probe for detecting metal ions, which is prepared by mixing the carbon nanodot solution prepared by the present invention with a phosphate buffer solution.
Example four
1. Preparation of carbon nanodot solution
The preparation process of this step is the same as in example one.
2. Preparation of fluorescent probe for detecting amino acid
The preparation process of this step is the same as example two.
3. Colorimetric response of probes to glutathione
To illustrate the role of the probe in glutathione colorimetric detection, 0-21. mu.M glutathione standard solution was added to the probe solution in sequence and the change in absorbance was recorded. The absorbance gradually increased with increasing glutathione concentration. FIG. 10 is a linear change curve diagram of colorimetric response of a probe prepared by mixing a carbon nano-dot prepared by the present invention with a phosphate buffer solution and then adding 10 μ M mercury ion standard solution to detect amino acids to glutathione.
4. Selectivity of colorimetric probes for amino acids
In order to evaluate the selectivity of the probe for colorimetric detection of glutathione, a series of amino acids, namely cysteine (Cys), phenylalanine (Phe), glycine (Gly), arginine (Arg), proline (Pro), tryptophan (Trp), serine (Ser), threonine (Thr) and histidine (His), are subjected to colorimetric selectivity experiments in sequence under the same conditions, and the colorimetric detection is similar to the fluorescence detection in example II, so that the colorimetric probe is selective for glutathione. Fig. 11 is a selective colorimetric detection diagram of Glutathione (GSH), cysteine (Cys), phenylalanine (Phe), glycine (Gly), arginine (Arg), proline (Pro), tryptophan (Trp), serine (Ser), threonine (Thr), and histidine (His) for preparing a probe for detecting amino acids by mixing the carbon nano-dots prepared by the present invention with a phosphate buffer solution and then adding 10 μ M of mercury ion standard solution.
EXAMPLE five
1. Preparation of carbon nanodot solution
The preparation process of this step is the same as in example one.
2. Detection of mercury ions in actual water sample
And (2) fully mixing 10 mu l of carbon dot stock solution with 2ml of different water samples (lake water and tap water), adding into a cuvette, then respectively adding mercury ion standard solutions with different concentrations, and finally detecting that the recovery rates of mercury ions are 96.0-104.5% and 95.4-103.7% respectively through a fluorescence mode and a colorimetric mode, wherein the results show that the method can be used for real-time detection of mercury ions in an environmental sample. FIG. 12 is a real-time change diagram of the color of the probe for detecting metal ions prepared by mixing the carbon nano-particles prepared by the invention with phosphate buffer solution with the addition of mercury ions, wherein the solution color gradually changes from purple to colorless.
EXAMPLE six
1. Preparation of carbon nanodot solution
The preparation process of this step is the same as in example one.
2. Detection of glutathione in actual water sample
Fully mixing 10 mu l of carbon dot stock solution with 2ml of different water samples (lake water and tap water), adding the mixture into a cuvette, adding 10 mu M of mercury ion standard solution, then respectively adding glutathione standard solutions with different concentrations, and finally detecting that the recovery rates of glutathione are 94.8-104.0% and 95.2-103.6% respectively through a fluorescence mode and a colorimetric mode, wherein the results show that the method can be used for real-time detection of glutathione in environmental samples. FIG. 13 shows that the color of the probe solution prepared by mixing the carbon nano-dots with the phosphate buffer solution and then adding 10. mu.M of the mercury ion standard solution to detect the amino acid changes in real time with the addition of glutathione, the color of the solution changes in real time with the addition of glutathione, and the color of the solution gradually changes from colorless to purple. FIG. 14 is a comparison graph of the interfering fluorescence response curves of the dual-mode probe prepared according to the present invention for removing copper ions.
EXAMPLE seven
The carbon nanodot solution was prepared in a similar manner to example one, except that the amount ratio of citric acid to urea was 1:2.
Example eight
The carbon nanodot solution was prepared in a similar manner to example one, except that the amount ratio of citric acid to urea was 1: 2.5.
Example nine
The carbon nanodot solution was prepared in a similar manner to example one, except that the amount ratio of citric acid to urea was 1: 3.5.
Example ten
The carbon nanodot solution was prepared in a similar manner to example one, except that the amount ratio of citric acid to urea was 1: 4.
EXAMPLE eleven
The preparation method of the carbon nanodot solution is similar to that of the first embodiment, except that the ultrasonic treatment time is 12min, the solution after complete ultrasonic treatment is transferred to a polytetrafluoroethylene-lined 50ml reaction kettle, and the solution is heated at 165 ℃ for 11 h.
Example twelve
The preparation method of the carbon nano-dot solution is similar to that of the first embodiment, except that the ultrasonic time is 18min, the solution after complete ultrasonic treatment is transferred to a 50ml reaction kettle with a polytetrafluoroethylene lining, and the solution is heated for 9h at 195 ℃.
EXAMPLE thirteen
The carbon nanodot solution was prepared in a similar manner to example one, except that it was centrifuged at 8000rpm for 11 min.
Example fourteen
The carbon nanodot solution was prepared in a similar manner to example one, except that it was centrifuged at 12000rpm for 9 min.
Example fifteen
The carbon nanodot solution was prepared in a similar manner to example one except that the centrifuged solution was filtered through a 0.2 μ M filter and then dialyzed with a cellulose ester dialysis bag for 23 hours.
Example sixteen
The carbon nanodot solution was prepared in a similar manner to example one except that the centrifuged solution was filtered through a 0.24 μ M filter and then dialyzed for 25 hours using a cellulose ester dialysis bag.
Example seventeen
A probe for detecting metal ions was prepared in a similar manner to the first example, except that 8. mu.l of the stock solution of carbon dots was mixed with 1.5ml of phosphate buffer solution and added to the cuvette to prepare a probe solution.
EXAMPLE eighteen
A probe for detecting metal ions was prepared in a similar manner to example one, except that 12. mu.l of the stock solution of carbon dots was mixed well with 2.5ml of phosphate buffer solution and added to a cuvette to prepare a probe solution.
Example nineteen
The preparation method of the amino acid detecting probe is similar to the embodiment, except that the dosage of the carbon dot stock solution is 8 mu l, the dosage of the phosphate buffer solution is 1.5ml, and the dosage of the mercury ion standard solution is 8 mu M.
Example twenty
The preparation method of the amino acid detecting probe is similar to the embodiment, except that the dosage of the carbon dot stock solution is 12 mu l, the dosage of the phosphate buffer solution is 2.5ml, and the dosage of the mercury ion standard solution is 12 mu M.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.

Claims (9)

1. The carbon nanodot is characterized in that citric acid and urea are dissolved in formamide and mixed, and the carbon nanodot is prepared by a hydrothermal method, wherein the mass ratio of the citric acid to the urea is 1: 2-4.
2. The carbon nanodot as claimed in claim 1, wherein the heating temperature is 165-.
3. The novel bimodal probe prepared from the fluorescent carbon nanodots as claimed in any one of claims 1 to 2, wherein the preparation method comprises the following steps:
(1) fully mixing urea and anhydrous citric acid, taking formamide as a solvent, then adding the mixture into a reaction kettle, and carrying out hydrothermal reaction to obtain a purple black solution;
(2) cooling, centrifuging, filtering and dialyzing the solution obtained in the step (1) to obtain a carbon nanodot solution;
(3) and mixing the carbon nanodot solution with a phosphate buffer solution to prepare a probe for detecting metal ions, or mixing the carbon nanodot solution with the phosphate buffer solution and then adding a mercury ion standard solution to prepare the probe for detecting amino acids.
4. The novel bimodal probe as claimed in claim 3, wherein the amount ratio of citric acid to urea in step (1) is 1:2-4, ultrasonic treatment is performed for 12-18min before hydrothermal reaction, the solution after ultrasonic treatment is transferred to a reaction kettle with a polytetrafluoroethylene lining, and heating is performed at 165-195 ℃ for 9-11 h.
5. The novel bimodal probe as claimed in claim 3, wherein the centrifugation in step (2) is carried out at 8000-.
6. The novel bimodal probe as claimed in claim 3, wherein the preparation method of the probe for detecting metal ions in step (3) is as follows: 8-12 mul of carbon dot stock solution and 1.5-2.5ml of phosphate buffer solution are fully mixed and added into a cuvette to prepare a probe solution.
7. The novel bimodal probe as claimed in claim 3, wherein the method for preparing the probe for detecting amino acid in step (3) comprises: and (3) fully mixing the carbon dot stock solution and a phosphate buffer solution, adding the mixture into a cuvette, adding 10 mu M of mercury ion standard solution, and finally preparing a probe solution for detecting amino acid.
8. The novel bimodal probe for detecting metal ions, prepared in the step (3), is used for fluorescence detection and colorimetric detection of mercury ions, and the bimodal probe for detecting amino acid is used for fluorescence detection and colorimetric detection of glutathione.
9. The dual-mode probe prepared by the preparation method of any one of 3-8 is applied to the selection of metal cations, fluorescence detection and colorimetric detection, and the selection of amino acids, fluorescence detection and colorimetric detection.
CN202111244410.7A 2021-10-25 2021-10-25 Carbon nanodot, dual-mode probe prepared based on carbon nanodot and application of dual-mode probe Pending CN114229827A (en)

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