CN110361371B - Fluorescence sensor based on nitrogen-doped carbon dots and catalytic oxidation reaction, and construction method and application thereof - Google Patents
Fluorescence sensor based on nitrogen-doped carbon dots and catalytic oxidation reaction, and construction method and application thereof Download PDFInfo
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Abstract
The invention discloses a method for detecting copper ions by a fluorescent sensor, which takes nitrogen-doped carbon dots as a fluorescent probe, utilizes the copper ions to specifically catalyze and oxidize cysteine to form cystine and hydrogen peroxide, further catalyzes and oxidizes the hydrogen peroxide to generate hydroxyl radicals to quench the fluorescence of the nitrogen-doped carbon dots, and constructs the fluorescent sensor for detecting the copper ions based on the method. When copper ions with different concentrations are added for reaction, the fluorescence intensity of the fluorescence sensor is gradually quenched, so that the fluorescence sensor can be used for quantitative detection of the copper ions; meanwhile, the fluorescence sensor can also be applied to the determination of the content of copper ions in human serum. The method has the advantages of simple operation, wide applicability, quick reaction and the like, and can carry out high-selectivity and high-sensitivity quantitative analysis on the target.
Description
Technical Field
The invention relates to a fluorescence sensor and application thereof, belonging to the fields of analytical chemistry and nanotechnology.
Background
Many transition metal ions play a crucial role in human life activities. Among these metal ions, copper ion (cu (ii)) is a divalent cation indispensable to the human body, which helps the organism to form red blood cells and acts as a neurotransmitter of physiological functions, and also acts as a catalytic cofactor of various metalloenzymes, including superoxide dismutase, cytochrome c oxidase, tyrosinase, and nuclease. When the copper content in a human body is abnormal, the copper content has obvious negative effects on the health of the human body, and excessive administration of Cu (II) can cause oxidative stress and neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, Menkes and Wilson's disease. Furthermore, cu (ii) has become a major heavy metal contaminant in the environment due to a wide range of industrial and agricultural applications. Since it is not biodegradable and will eventually accumulate in the human body through the food chain, it is essential to develop an accurate and sensitive strategy for detecting cu (ii) for human health.
The traditional cu (ii) detection means are based on graphite flame atomic absorption spectroscopy, surface plasmon resonance sensors, inductively coupled plasma mass spectrometry, inductively coupled plasma atomic emission spectroscopy, plasma resonance rayleigh scattering spectroscopy, etc. These methods all have a relatively high sensitivity, but their detection process is relatively complicated and requires expensive instruments and trained personnel. In recent years, fluorescent sensors have received increasing attention because of their advantages such as being non-destructive, cost-effective, and high sensitivity.
A fluorescence sensor is a molecular device that is sensitive to a specific analyte and can selectively interact with it to convert its concentration change into an optical signal or an electrical signal change output for detection. The fluorescence sensor technology adopts fluorescent groups, including inorganic materials such as up-conversion nano materials and quantum dots, and organic materials such as green fluorescent protein, red fluorescent protein or fluorescent dye to detect a target object. In recent years, fluorescent sensors have been widely used in the detection of target substances in some biological samples.
Nitrogen-doped carbon dots (N-CDs) have become a new photoluminescent inorganic material with low cost, low toxicity and good biocompatibility, and are widely used for fluorescence sensing and detection of bioactive substances. Patent CN 109321237 a discloses a nitrogen-sulfur double-doped carbon dot, a preparation method and an application thereof, comprising the following steps: taking papermaking waste sodium lignin sulfonate and cysteine as raw materials, placing the raw materials in a hydrothermal reaction kettle, carrying out hydrothermal reaction,after the reaction was completed, the reaction mixture was cooled to room temperature, and the obtained black product was centrifuged. Filtering the collected solution part by using a filter membrane, wherein the brown yellow solution obtained after filtering is a blue fluorescent carbon quantum dot solution, and the obtained carbon quantum dot can be used for Fe in water3+The analysis and detection of (3); the invention discloses a nitrogen-doped carbon dot and a preparation method and application thereof, and the preparation method comprises the following steps: placing urea and citric acid as raw materials in a hydrothermal reaction kettle for hydrothermal reaction, cooling to room temperature after the reaction is finished, and adding ethanol into the obtained product for centrifugal purification to obtain the product. The obtained nitrogen-doped carbon dots can be used for analyzing and detecting Cu (II) in serum. Compared with the method, the method has the following advantages: (1) only nitrogen doping in the carbon dots is required for detection. It is not necessary to dope both elements. (2) The operation steps for manufacturing the nitrogen-doped carbon dots are simple, the time is shorter, and only 8 hours are needed. (3) The detection range of the method for detecting the copper ions is wide and is 0.05 mu M-100 mu M. (4) The components in the serum are complex, and the method has better selectivity.
The invention constructs a fluorescence sensor suitable for detecting copper ions by utilizing the fact that copper ions can specifically catalyze and oxidize cysteine to form cystine and hydrogen peroxide and further catalyze and oxidize the hydrogen peroxide to generate hydroxyl radicals to quench fluorescence of nitrogen-doped carbon dots, and applies the fluorescence sensor to detection of the content of the copper ions in human serum.
Disclosure of Invention
Aiming at the problem of poor selectivity of a copper ion chelation quenching type fluorescence sensor, the invention provides a fluorescence sensor based on catalytic oxidation reaction, cysteine in the fluorescence sensor can be specifically catalyzed and oxidized by copper ions to form cystine and hydrogen peroxide, and the cysteine is further catalyzed and decomposed by the hydrogen peroxide to generate hydroxyl radicals to destroy nitrogen-doped carbon points to quench fluorescence of the fluorescence sensor. When copper ions with different concentrations are added for reaction, the blue fluorescence of the fluorescence sensor can be gradually quenched, so that high-selectivity and high-sensitivity quantitative detection of the copper ions is realized. Meanwhile, the fluorescence sensor can also be applied to the quantitative detection of copper ions in human serum.
In order to achieve the purpose, the invention adopts the following technical scheme:
a fluorescence sensor is constructed by taking nitrogen-doped carbon dots as a fluorescence probe, specifically catalyzing and oxidizing cysteine to form cystine and hydrogen peroxide by utilizing copper ions, and further catalyzing and oxidizing the hydrogen peroxide to generate hydroxyl radicals to quench fluorescence of the nitrogen-doped carbon dots.
The nitrogen-doped carbon dots are obtained by performing hydrothermal reaction on citric acid and urea serving as a carbon source and a nitrogen source and water serving as a solvent at 160 ℃ for 8 hours and then adding ethanol for centrifugal purification; wherein the mass ratio of the citric acid to the urea is 1: 1; the mass-volume ratio of the urea to the water is 1:15 g/mL; the volume ratio of water to ethanol is 1: 3.
The construction method of the fluorescence sensor comprises the following steps: stirring and mixing the nitrogen-doped carbon dot solution and the cysteine solution according to the volume ratio of 1:1 to prepare the fluorescent sensor; wherein, the concentration of the nitrogen-doped carbon dot solution is 50 mug/mL, and the concentration of the cysteine solution is 1.2 mM.
The application of the fluorescence sensor in quantitative detection of copper ions comprises the following steps:
and respectively adding copper ion standard solutions with gradient concentrations into the fluorescence sensors, mixing and reacting for 25 min, measuring fluorescence intensity by using a fluorescence spectrophotometer, and drawing a standard curve of the fluorescence intensity along with the change of the copper ion concentration.
The application of the fluorescence sensor in human serum detection comprises the detection of the content of copper ions in human serum, and specifically comprises the following steps:
and adding human serum collected by a hospital into the fluorescence sensor, reacting for 25 min, and measuring the fluorescence intensity by using a fluorescence spectrophotometer to realize the measurement of the copper ion content in the human serum.
The invention has the beneficial effects that:
(1) the raw material nitrogen-doped carbon dots utilized in the invention are a luminescent material which is simple and convenient to synthesize and has excellent fluorescence performance, and the raw materials are convenient and easy to obtain.
(2) The fluorescence sensor of the invention responds to copper ions with different concentrations to show the change of fluorescence quenching, has the advantage of good selectivity compared with a copper ion chelating quenching type fluorescence sensor, and has the advantage of simple and convenient operation.
(3) The fluorescent sensor is suitable for quantitative detection of copper ions in human serum.
Drawings
FIG. 1 is a schematic diagram of the fluorescence sensor of the present invention for detecting the content of copper ions.
Fig. 2 is a characterization map of nitrogen-doped carbon dots according to the present invention: (A) a transmission electron microscope image; (B) ultraviolet-visible absorption spectrogram; (C) fluorescence excitation and emission spectra; (D) fourier infrared spectrogram.
FIG. 3 is a graph showing the change of fluorescence intensity of the fluorescence sensor according to the present invention in response to different concentrations of copper ions.
FIG. 4 shows a selective study of the fluorescence sensor of the present invention for copper ion detection.
Detailed Description
In order to make the present invention more comprehensible, the technical solutions of the present invention are further described below with reference to specific embodiments, but the present invention is not limited thereto.
Example 1: synthesis of nitrogen-doped carbon dots
Firstly, 1 g of citric acid and 1 g of urea are ultrasonically dissolved in 15 mL of water, when the solution is clear and transparent, the solution is transferred into a polytetrafluoroethylene high-pressure reaction kettle with the volume of 50 mL, heating at 160 ℃ for reaction for 8 h, adding ethanol with the volume three times that of the obtained product after the reaction kettle is cooled to room temperature, uniformly mixing, and centrifuging at 5000rpm to remove liquid, repeatedly washing with ethanol for three times, and vacuum drying the obtained solid in a vacuum oven to obtain solid crystal of the nitrogen-doped carbon point (the related characteristics of the nitrogen-doped carbon point are shown in figure 2, wherein A is a transmission electron microscope image of the nitrogen-doped carbon point and shows that the particle size distribution is 2-3 nm, B is an ultraviolet visible absorption spectrogram of the nitrogen-doped carbon point, C is a fluorescence excitation and fluorescence emission spectrogram of the nitrogen-doped carbon point, and D is an Fourier external spectrogram of the nitrogen-doped carbon point).
Example 2: construction of the fluorescence sensor and specific response of copper ions
And adding 1.2 mM cysteine solution into 50 mu g/mL nitrogen-doped carbon dot solution s according to the volume ratio of 1:1 to prepare the fluorescent sensor.
The obtained fluorescence sensor was added with a copper ion standard solution of a gradient concentration (the concentration of the copper ion standard solution was 0.05, 0.1, 0.5, 1, 2, 4, 6, 9, 15, 20, 25, 40, 60, 80, 100. mu.M in this order), reacted for 25 min, and the fluorescence intensity was measured with a fluorescence spectrophotometer and a fluorescence intensity change curve was plotted. (for example, FIG. 2 is a graph showing the change of fluorescence intensity after reaction and different concentrations of copper ions).
Example 3: the method for detecting the content of copper ions in human serum comprises the following specific steps
And adding human serum collected by a hospital into the fluorescence sensor, reacting for 25 min, measuring the fluorescence intensity by using a fluorescence spectrophotometer, and recording data. The detection result shows that the content of the copper ions in the human serum is 1.63 mu M.
Example 3: selectivity of fluorescence sensor for copper ion detection
Interfering substances are respectively added into the fluorescence sensor, the concentration of copper ions is 20 mu M, and the concentration of other interfering substances is 200 mu M. As shown in fig. 4, blank, sodium ion, potassium ion, magnesium ion, zinc ion, calcium ion, aluminum ion, ferrous ion, iron ion, bovine serum albumin, lysine, vitamin B2, glucose, glutathione, ascorbic acid and copper ion are respectively from left to right. The corresponding fluorescence intensity change is shown in fig. 4, except that copper ions quench the fluorescence intensity of the fluorescence sensor, other interfering substances do not affect the fluorescence intensity. The result shows that the fluorescence sensor prepared by the method has better selectivity for detecting copper ions.
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
Claims (6)
1. A fluorescence sensor based on nitrogen-doped carbon dots and catalytic oxidation reaction is characterized in that: constructing a fluorescent sensor for detecting copper ions by taking nitrogen-doped carbon dots as a fluorescent probe and taking hydroxyl radicals generated by the reaction of cysteine and copper ions as a quencher; the nitrogen-doped carbon dots are obtained by performing hydrothermal reaction on citric acid and urea serving as a carbon source and a nitrogen source and water serving as a solvent at 160 ℃ for 8 hours, and then adding ethanol for centrifugal purification; wherein the mass ratio of the citric acid to the urea is 1: 1; the mass-volume ratio of the urea to the water is 1:15 g/mL; the volume ratio of water to ethanol is 1: 3.
2. A method of constructing a fluorescence sensor according to claim 1, wherein: the construction method of the fluorescence sensor comprises the following steps: and mixing the nitrogen-doped carbon dot solution and the cysteine solution according to the volume ratio of 1:1 to prepare the fluorescent sensor for detecting the copper ions.
3. The method of constructing a fluorescence sensor according to claim 2, wherein: the concentration of the nitrogen-doped carbon dot solution is 50 mu g/mL, and the concentration of the cysteine solution is 1.2 mM.
4. Use of a fluorescence sensor according to claim 1 for the quantitative detection of copper ions, characterized in that: the application method comprises the following steps: and respectively adding copper ion standard solutions with series concentrations into the fluorescence sensor, mixing and reacting for 25 min, measuring fluorescence intensity by using a fluorescence spectrophotometer, and drawing a standard curve of the fluorescence intensity along with the change of the copper ion concentration.
5. Use of the fluorescence sensor according to claim 4 for the quantitative detection of copper ions, characterized in that: the method is used for detecting the content of copper ions in human serum.
6. The use of the fluorescence sensor according to claim 5 for the quantitative detection of copper ions, characterized in that: the method is used for detecting the content of copper ions in human serum, and comprises the following steps: and adding human serum collected by a hospital into the fluorescence sensor, reacting for 25 min, and measuring the fluorescence intensity by using a fluorescence spectrophotometer to realize the measurement of the copper ion content in the human serum.
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