CN112763438A - Application of carbon dot peroxidase CDs @ NC in detection of D-alanine and D-proline - Google Patents
Application of carbon dot peroxidase CDs @ NC in detection of D-alanine and D-proline Download PDFInfo
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Abstract
The invention provides an application of carbon dot peroxidase CDs @ NC in detection of D-alanine and D-proline, wherein D-Ala and D-Pro can generate H in the presence of DAAO2O2In H2O2And the colorless TMB is changed into blue TMB under the combined action of the fluorescent dye and CDs @ NC, and an obvious absorption peak appears at 652 nm on an ultraviolet-visible spectrum, and the rapid detection of the D-Ala and the D-Pro can be realized based on the change of the ultraviolet absorption peak intensity along with the concentration of the D-Ala and the D-Pro. The method does not need to use expensive large instruments, and has wide application range and good environmental adaptability. The nano enzyme involved in the method is a carbon material, has low price and good stability, and can be prepared in large scale.
Description
Technical Field
The invention relates to application of carbon-point peroxidase CDs @ NC in detection of D-alanine and D-proline, and belongs to the technical field of non-noble metal catalysts in electrocatalysis.
Background
Researches show that the survival rate of the gastric cancer after late treatment is far lower than that of early treatment, so that the early discovery and the timely treatment of the gastric cancer have important significance. However, most of the early gastric cancer patients have no specific symptoms, which causes difficulty in timely diagnosis. Conventional diagnostic methods for early gastric cancer include endoscopy, biopsy, and blood examination. However, these methods are complex, time consuming, dependent on well-equipped laboratory facilities and can be traumatic to human tissue, and thus there is a strong need for a convenient, rapid, non-invasive method for gastric cancer detection.
It is reported that the concentrations of D-alanine (D-Ala) and D-proline (D-Pro) in gastric juice and saliva of early gastric cancer patients are 126.3-285.3. mu.M and 46.1-114.5. mu.M, respectively, which are much higher than those of healthy persons (7.3-18.3. mu.M and 5.4-7.8. mu.M, respectively). Therefore, accurate monitoring of the concentrations of D-Ala and D-pro is of great importance for the diagnosis of gastric cancer. At present, many detection methods have been reported, including chromatography, electrochemiluminescence, electrochemical biosensor method, etc. However, colorimetric detection of D-Ala and D-Pro is rarely reported. Previous studies have reported that D-amino oxidase (DAAO) can selectively oxidize D-Ala and D-Pro to H2O2In peroxidase and H2O2The colorless TMB changes to blue TMB. Therefore, D-Ala and D-Pro can be used as potential biomarkers for gastric cancer detection.
The nano enzyme is used as a combination of mimic enzyme and nano material, has been rapidly developed in the last years, is widely applied in the fields of analysis sensing and biomedicine, and has lower production cost, easier mass production and better stability compared with natural enzyme. Among them, carbon-based nanoenzymes are attractive due to their high stability, low cost and good biocompatibility. Carbon dots, a typical carbon material, has attracted considerable attention due to high stability, low toxicity and high biocompatibility, and in previous studies, it has been found that carbon dots have intrinsic peroxidase activity. However, the factors such as easy agglomeration, uneven size and morphology and the like reduce the peroxidase activity of the carbon dots. ZIF-8 is a typical metal-organic framework material, has a stable framework and uniform internal pore size (1.9 nm), and glucose molecules can be loaded into pores of the ZIF-8 in a liquid phase and carbonized at 200 ℃ to generate carbon dots. In particular, the peroxidase activity of the carbon material can also be improved by a nitrogen doping method. However, the synergistic effect of carbon dots and nitrogen-doped carbon to increase peroxidase activity has not been previously reported.
Disclosure of Invention
Aiming at the problem that the current carbon material peroxidase is low in activity, ZIF-8 is used as a template, glucose is used as a carbon source, carbon dots with uniform size are obtained at 200 ℃, then the carbon dots are further carbonized at high temperature to obtain nitrogen-doped carbon-coated carbon dots (CDs @ NC) which are used as a novel peroxidase, and then the peroxidase activity of the novel nano-enzyme is utilized to rapidly and efficiently detect D-Ala and D-Pro. The detection mechanism is as follows: D-Ala and D-Pro produce H in the presence of DAAO2O2And in H2O2And CDs @ NC, colorless TMB can be changed into blue TMB, and an obvious absorption peak is shown at 652 nm on an ultraviolet-visible spectrum, and the detection of D-Ala and D-Pro can be realized based on the change of the intensity of the ultraviolet absorption peak along with the concentration of D-Ala and D-Pro.
The invention is realized by the following technical scheme:
application of carbon dot peroxidase CDs @ NC in detection of D-alanine and D-proline. D-Ala and D-Pro produce H in the presence of DAAO2O2In H2O2And CDs @ NC, and the colorless TMB is changed into blue TMB, and an obvious absorption peak appears at 652 nm on the ultraviolet-visible spectrum, and the detection of D-Ala and D-Pro can be realized based on the change of the ultraviolet absorption peak intensity along with the concentration of D-Ala and D-Pro.
Further, the preparation method of the CDs @ NC comprises the following steps:
preparation of ZIF-8: dissolving 4 g of dimethylimidazole in 60 mL of methanol solution; 1.68 g of Zn (NO)3)2·6H2Dissolving O into 20 mL of methanol solution, adding the solution, and violently stirring at room temperature for 1 h; incubating the mixture at room temperature for 24 h without stirring; centrifugally washing and drying to obtain ZIF-8;
② G @ ZIF-8 preparation: immersing 200 mg ZIF-8 powder in glucose solution, stirring at room temperature for 6H, centrifuging to separate white precipitate, and treating with EtOH/H2O = 9: 1, and 6Drying overnight at 0 ℃ under vacuum;
preparing CDs @ ZIF-8: heating the G @ ZIF-8 powder to 200 ℃, preserving heat, and cooling to room temperature to obtain CDs @ ZIF-8;
preparing CDs @ NC: and heating the powder of CDs @ ZIF-8 to 900 ℃ and preserving heat to obtain CDs @ NC.
Further, the concentration of the glucose solution is 1-3 mM, and the solution is EtOH/H2O = 9: 1 in volume ratio.
Further, the G @ ZIF-8 powder is heated to 200 ℃ in an atmosphere of N2The heating rate is 5 ℃ for min-1The heat preservation time is 3 hours.
Further, the CDs @ ZIF-8 powder is heated to 900 ℃ in an atmosphere of N2The heating rate is 5 ℃ for min-1The heat preservation time is 3 hours.
Advantageous effects
The invention discloses an application of carbon dot peroxidase CDs @ NC in detection of D-alanine and D-proline, which comprises the following steps:
(1) the method does not need to use expensive large instruments, and has wide application range and good environmental adaptability.
(2) The nano enzyme involved in the method is a carbon material, has low price and good stability, and can be prepared in large scale.
(3) The method can detect the concentrations of D-alanine (D-Ala) and D-proline (D-Pro), and has important significance for further using the system in the detection of human saliva samples, causing no harm to human bodies, having simpler operation and performing noninvasive diagnosis on early gastric cancer patients.
Drawings
Fig. 1 (a) transmission electron micrograph image of nitrogen-doped carbon-coated carbon dots; (B) a high resolution transmission electron micrograph of nitrogen-doped carbon-coated carbon dots; (C) a lattice fringe image of carbon dots;
FIG. 2 (A) is a graph showing UV-VIA absorption spectra of D-Ala added to the detection system at different concentrations, wherein the concentrations of D-Ala are 0, 10, 20, 30, 50, 100, 200, 300, 400, 500, 600, 800 and 1000. mu.M from top to bottom; (B) the relationship between the absorbance of the reaction system at 652 nm and the concentration of D-Ala is shown in the linear range;
FIG. 3 (A) is a graph of the UV-VIS absorption spectra of the detection system after adding D-Pro of different concentrations, wherein the concentrations of D-Pro are 0, 10, 20, 30, 40, 50, 100, 150, 200, 300, 400, 600, 800 and 1000. mu.M from top to bottom; (B) the relationship between the absorbance of the reaction system at 652 nm and the concentration of D-Pro is shown in the inset as the linear range;
FIG. 4 (A) anti-interference capability test of detection system; (B) testing of human saliva samples, samples 1, 2, 3 from healthy persons; samples 4, 5, 6 were from gastric cancer patients;
FIG. 5 shows the absorbance of each sample, from top to bottom, as CDs @ NC-3, CDs @ NC-2, CDs @ NC-1, ZIF-8-900+ CDs, ZIF-8-900, and CDs @ NC-1, where the glucose concentration is 1 mM; CDs @ NC-2 indicates a glucose concentration of 2 mM; CDs @ NC-3 indicates a glucose concentration of 3 mM; CDs denotes the carbon spots obtained by etching the prepared CDs @ ZIF-8 by immersion in a 1 mM KOH solution and then extracting by filtration with diethyl ether; ZIF-8-900 denotes a material directly heated to 900 ℃ without adding glucose; ZIF-8-900+ CDs are materials obtained by mechanically mixing ZIF-8-900 with CDs.
Detailed Description
The following examples are given for the detailed implementation and specific operation of the present invention, but the scope of the present invention is not limited to the following examples.
Example 1
(1) Preparation and characterization of nitrogen-doped carbon-coated carbon dots:
preparation of ZIF-8. Dissolving 4 g of dimethylimidazole in 60 mL of methanol solution; 1.68 g of Zn (NO)3)2·6H2Dissolving O into 20 mL of methanol solution, adding the solution, and violently stirring at room temperature for 1 h; incubating the mixture at room temperature for 24 h without stirring; and (4) centrifugally washing and drying.
② G @ ZIF-8. 200 mg of ZIF-8 powder was immersed in a glucose (G) solution (1-3 mM EtOH/H)2O = 9: 1) stirring at room temperature for 6H, centrifuging to separate white powder, and adding EtOH/H2O = 9: 1 was washed three times and finally dried overnight at 60 c under vacuum.
Preparing CDs @ ZIF-8. Putting the G @ ZIF-8 powder into a tube furnace, heating the powder from room temperature to 200 ℃ under the condition of nitrogen, and preserving the heat for 3 hours at 200 ℃, wherein the heating rate is 5 ℃ for min-1. After cooling to room temperature, CDs @ ZIF-8 was obtained.
And fourthly, preparing CDs @ NC. Transferring the CDs @ ZIF-8 powder into a tube furnace, heating the powder from room temperature to 900 ℃ under the condition of nitrogen, and keeping the temperature for 3 hours at the heating rate of 5 ℃ for min-1And obtaining the nitrogen-doped carbon-coated carbon dots.
And (3) characterizing the prepared nano enzyme by using a transmission electron microscope and an ultraviolet-visible absorption spectrum. From the characterization result of the transmission electron microscope in FIG. 1, it can be seen that the size of the prepared nanoenzyme is about 250 nm. At high resolution, many small black dots with a size of about 1.9 nm and a lattice fringe of carbon dots (d =0.21 nm) were visible, indicating that we successfully produced nitrogen-doped carbon-coated carbon dots.
Preparing CDs @ NC materials with different concentrations of glucose content respectively, preparing different materials by using different heating temperatures, and comparing the absorbance of the materials; see FIG. 5 absorbance for different samples, CDs @ NC-1 indicates a glucose concentration of 1 mM; CDs @ NC-2 indicates a glucose concentration of 2 mM; CDs @ NC-3 indicates a glucose concentration of 3 mM; CDs denotes the carbon spots obtained by etching the prepared CDs @ ZIF-8 by immersion in a 1 mM KOH solution and then extracting by filtration with diethyl ether; ZIF-8-900 denotes a material directly heated to 900 ℃ without adding glucose; ZIF-8-900+ CDs are materials obtained by mechanically mixing ZIF-8-900 with CDs. The different absorbance represents the different peroxidase-like activity of the material, and the higher the absorbance, the better the peroxidase-like activity of the material.
(2) Determination of D-Ala (or D-Pro): first, 10. mu.L of DAAO (10U mL)-1) Incubation of a mixture of solutions, 20 μ L of different concentrations of D-Ala (or D-Pro) solutions and 170 μ L of PBS buffer (pH =6.8) at 37 ℃ for 45 min yielded H2O2。Then, 50. mu.L of TMB (5mM) solution and 20. mu.L of CDs @ NC (0.5 mg mL) were added to the above solution-1) And 730. mu.L of acetic acid-bSodium buffer (pH =4.0, 0.2M). The incubation was then carried out at 30 ℃ for 30 minutes and the UV-visible absorption spectra were collected.
In FIG. 2 (A), the UV-VIS absorption spectra of the detection system added with D-Ala at different concentrations are shown in the order of 0, 10, 20, 30, 50, 100, 200, 300, 400, 500, 600, 800 and 1000. mu.M from top to bottom. (B) The reaction system has a relationship between the absorbance at 652 nm and the concentration of D-Ala, the linear range is shown in the inset. FIG. 3 (A) is a graph of the UV-VIS absorption spectra of the detection system after adding D-Pro of different concentrations, wherein the concentrations of D-Pro are 0, 10, 20, 30, 40, 50, 100, 150, 200, 300, 400, 600, 800 and 1000. mu.M from top to bottom; (B) the reaction system has the relationship between the absorbance at 652 nm and the concentration of D-Pro, and the linear range is shown in the inset. The linear range for D-Ala was found to be: 20-400 μ M, detection limit 20 μ M. The linear range of D-Pro is: 20-300 μ M, detection limit 20 μ M.
FIG. 4 shows the anti-interference ability test and the detection results of the detection system, the content of D-Ala (or D-Pro) in saliva of early gastric cancer patients is above the detection limit of our detection system, and the content of saliva of healthy people is below the detection limit of our detection system.
The substances which possibly influence a detection system and exist in the saliva are detected, and researches show that the substances do not influence the detection system after being added, so that the method is proved to have better selectivity.
(3) Determination of human saliva samples: we measured saliva for three healthy people and three patients with gastric cancer. Human saliva samples were taken directly for testing. The assay was performed as for D-Ala (or D-Pro). The detection result shows that saliva of healthy people does not develop color in the detection system, and saliva of gastric cancer patients develops color in the detection system, which has important significance for diagnosis of gastric cancer patients.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Claims (6)
1. Application of carbon dot peroxidase CDs @ NC in detection of D-alanine and D-proline.
2. Use of the carbodotropic peroxidase CDs @ NC according to claim 1 for the detection of D-alanine and D-proline, characterized in that D-Ala and D-Pro produce H in the presence of DAAO2O2In H2O2And CDs @ NC, and the colorless TMB is changed into blue TMB, and an obvious absorption peak appears at 652 nm on the ultraviolet-visible spectrum, and the detection of D-Ala and D-Pro can be realized based on the change of the ultraviolet absorption peak intensity along with the concentration of D-Ala and D-Pro.
3. The application of the carbon dot type peroxidase CDs @ NC in the detection of D-alanine and D-proline according to claim 1, wherein the preparation method of the CDs @ NC is as follows:
preparation of ZIF-8: dissolving 4 g of dimethylimidazole in 60 mL of methanol solution; 1.68 g of Zn (NO)3)2·6H2Dissolving O into 20 mL of methanol solution, adding the solution, and violently stirring at room temperature for 1 h; incubating the mixture at room temperature for 24 h without stirring; centrifugally washing and drying to obtain ZIF-8;
② G @ ZIF-8 preparation: immersing 200 mg ZIF-8 powder in glucose solution, stirring at room temperature for 6H, centrifuging to separate white precipitate, and treating with EtOH/H2O = 9: 1, washing three times, and drying overnight at 60 ℃ under vacuum;
preparing CDs @ ZIF-8: heating the G @ ZIF-8 powder to 200 ℃, preserving heat, and cooling to room temperature to obtain CDs @ ZIF-8;
preparing CDs @ NC: and heating the powder of CDs @ ZIF-8 to 900 ℃ and preserving heat to obtain CDs @ NC.
4. The use of the carbodotropic peroxidase CDs @ NC according to claim 3 for the detection of D-alanine and D-prolineCharacterized in that the concentration of the glucose solution is 1-3 mM, and the solution is EtOH/H2O = 9: 1 in volume ratio.
5. The use of the carbon-point peroxidase CDs @ NC according to claim 3 for detecting D-alanine and D-proline, wherein the G @ ZIF-8 powder is heated to 200 ℃ in an atmosphere of N2The heating rate is 5 ℃ for min-1The heat preservation time is 3 hours.
6. The use of the carbon-point peroxidase CDs @ NC according to claim 3 for detecting D-alanine and D-proline, wherein the powder of CDs @ ZIF-8 is heated to 900 ℃ in an atmosphere of N2The heating rate is 5 ℃ for min-1The heat preservation time is 3 hours.
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Cited By (3)
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| CN113460993A (en) * | 2021-06-29 | 2021-10-01 | 湘潭大学 | Zinc-nitrogen modified dual-carbon catalytic material, preparation method thereof and application thereof in zinc-air battery |
| CN113493197A (en) * | 2021-07-22 | 2021-10-12 | 哈尔滨工程大学 | Preparation method of carbon dot/porous silicon dioxide nanoenzyme with peroxidase-like activity |
| CN114479113A (en) * | 2022-03-18 | 2022-05-13 | 中国农业科学院蔬菜花卉研究所 | Two-dimensional fluorescent MOFs composite material, preparation method and application thereof, and method for detecting iron ions by fluorescence |
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