CN115229177B - Preparation method and application of sodium humate-copper nanocluster - Google Patents
Preparation method and application of sodium humate-copper nanocluster Download PDFInfo
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- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6432—Quenching
Abstract
The invention relates to the technical field of analytical chemistry and fluorescent nano biological materials, in particular to a preparation method and application of sodium humate-copper nanoclusters. Comprising the following steps: step 1: weighing sodium humate, dissolving in ultrapure water to obtain a sodium humate solution as a protective agent, weighing copper salt, dissolving in ultrapure water to obtain a copper ion solution, mixing and stirring the two solutions, and regulating the pH value of the mixed solution to be 10-11; step 2: adding an ascorbic acid solution serving as a reducing agent into the mixed solution obtained in the step 1, placing the mixed solution on a constant-temperature magnetic stirrer, reacting for 10min at 60 ℃, standing the mixed solution after the reaction is finished, and cooling to room temperature; step 3: centrifuging the cooling mixed solution obtained in the step 2 in a high-speed centrifuge for 15min to obtain a pale yellow sodium humate-copper nanocluster solution at the upper layer, and then preserving the solution in a refrigerator at 4 ℃ in a dark place.
Description
Technical Field
The invention relates to the technical field of analytical chemistry and fluorescent nano biological materials, in particular to a preparation method and application of sodium humate-copper nanoclusters.
Background
Amino acids are an important evaluation index commonly used in chemical and biological systems that affect many chemical and biochemical reactions, especially the cancerous changes of cells. Studies have shown that the content of part of amino acids in tumor cells is abnormally changed, so that the detection amino acids can be used as a marker for cancer diagnosis. Tryptophan (Trp) has received much attention as one of the essential amino acids, because of its indispensable role in protein synthesis and biomedical metabolic regulation, and because its metabolic abnormality is closely related to the pathogenesis of cancer such as the occurrence of breast cancer, cervical cancer and liver cancer. In addition, common toxic metallic mercury ions (Hg 2+ ) The incidence of cancer is also being greatly focused due to its high toxicity and corrosiveness to cells. Therefore, there is an urgent need to develop a simple, environmentally friendly assay for tryptophan and Hg in biological systems 2+ And meanwhile, detection is carried out, so that the clinical early diagnosis of the cancer is realized and the kit is used for preventing the occurrence of the cancer.
Tryptophan and Hg 2+ Conventional measurement method of (2)The method mainly comprises an electrochemical method, a high performance liquid chromatography, a colorimetry method and the like. Although these methods are widely used, they have the disadvantages of expensive equipment, complicated operation, high cost, long time consumption and the like. Therefore, there is an urgent need to develop a rapid and simple analysis method for efficiently detecting tryptophan and Hg 2+ 。
The metal copper nanocluster fluorescent probe is a technology developed in recent years, and is favored by researchers because of small volume, small sample consumption, multiple repeated use, simple operation, high sensitivity and low cost. There are studies showing that it can also be used for detection of active substances in cells/tissues or internal environments. However, the copper nanoclusters prepared at present have the problems of easy aggregation, oxidation and the like, so that the storage stability is low, and fluorescence is easy to quench. In order to solve the two problems of low stability and complicated preparation of fluorescent copper nanoclusters, the structure to be selected comprises an aromatic ring and an alicyclic ring, the ring is provided with functional groups such as hydroxyl, carboxyl, quinolyl and the like, sodium humate which can perform complexation and redox with metal ions is used as a protective agent, and a one-pot chemical reduction method is adopted to prepare the copper nanoclusters with high stability. In addition, there are currently no bifunctional fluorescent copper nanoclusters that detect tryptophan and mercury ions.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention provides a preparation method and application of sodium humate-copper nanoclusters. In view of the convenience, greenness, excellent stability, good biocompatibility, fluorescence enhancement response to tryptophan and Hg of the sodium humate-protected copper nanocluster preparation method 2+ The copper nanocluster prepared by the method can be used for fluorescence enhanced tryptophan sensing and continuous Hg detection 2+ And (5) sensing.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
a method for preparing sodium humate-copper nanoclusters, comprising:
step 1: weighing sodium humate, dissolving in ultrapure water to obtain a sodium humate solution as a protective agent, weighing copper salt, dissolving in ultrapure water to obtain a copper ion solution, mixing and stirring the two solutions, and regulating the pH value of the mixed solution to be 10-11;
step 2: adding an ascorbic acid solution serving as a reducing agent into the mixed solution obtained in the step 1, placing the mixed solution on a constant-temperature magnetic stirrer, reacting for 10min at 60 ℃, standing the mixed solution after the reaction is finished, and cooling to room temperature;
step 3: centrifuging the cooling mixed solution obtained in the step 2 in a high-speed centrifuge for 15min to obtain a pale yellow sodium humate-copper nanocluster solution at the upper layer, and then preserving the solution in a refrigerator at 4 ℃ in a dark place.
Preferably, the copper ion solution in the step 1 is any one of copper nitrate, copper chloride, copper sulfate and copper acetate.
Preferably, the step 3 high speed centrifuge is operated at 12000 rpm/min.
Preferably, the mass ratio of the sodium humate to the ascorbic acid to the copper ions is 6:2:1.
The sodium humate-copper nanocluster can generate cyan fluorescence, the maximum excitation wavelength is 370nm, and the maximum emission wavelength is 458nm, and the sodium humate-copper nanocluster is used as a probe for fluorescence enhancement type tryptophan detection.
Preferably for continuous fluorescence quenching detection of mercury ions.
Preferably, the continuous measurement of mercury ions is realized by utilizing sodium humate-copper nanocluster-tryptophan, and weakening variation of fluorescence intensity at 458nm of fluorescence emission spectrum.
Compared with the prior art, the invention has the beneficial effects that:
(1) The sodium humate is used as a protective agent, and lone pair electrons in carboxyl and quinolyl groups contained in the sodium humate can be combined with Cu 2+ The hollow orbitals combine to form a chelate with a highly concentrated region, and then ascorbic acid is used as a reducing agent to reduce cupric ions into zero-valent copper atoms and monovalent copper ions, so that the sodium humate-protected copper nanoclusters are obtained.
The amino and carboxyl in tryptophan can form a stable hydrogen bond with carbonyl, methoxy and enol groups in sodium humate, so that aggregation of sodium humate-protected copper nanoclusters is induced, movement of electrons among sodium humate molecules is limited, fluorescence of the sodium humate is obviously enhanced, switch-enhanced tryptophan detection is further realized, and detection signal to noise ratio is obviously improved. And then mercury ions can form a non-luminous complex with amine groups in tryptophan through coordination, so that hydrogen bonds between tryptophan and copper nanoclusters are broken, a highly aggregated structure is broken, fluorescence is quenched, and continuous detection of mercury ions is realized.
(2) The method adopts one-pot reaction when preparing the sodium humate-copper nanocluster, which can promote the reaction to rapidly occur so as to shorten the reaction time and the operation flow;
(3) The method uses the sodium humate-protected copper nanocluster material as a probe, and changes the solution value into an optical signal, so that the method is convenient for naked eye observation and instrument measurement;
(4) The tryptophan detection method constructed by the invention is fluorescence enhancement type detection, the detection limit is 289nM, the detection sensitivity is high, the response range is wide, and the background interference is small;
(5) The method has the advantages that the reaction time is quick, the sodium humate-copper nanocluster can be obtained only in 10 minutes, and the method has the characteristics of environmental protection, economy, practicability and the like;
(6) The method provided by the invention realizes continuous detection of mercury ions, the detection limit is 159nM, the reaction time is 5min, and the detection time and the operation process are greatly shortened.
Drawings
The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate the invention and together with the embodiments of the invention, serve to explain the invention.
In the drawings:
FIG. 1 is a graph of copper nanoclusters irradiated with visible light and ultraviolet light;
FIG. 2 is a graph showing the effect of different reaction conditions and amounts of raw materials on copper nanocluster solutions;
FIG. 3 is a graph of the ultraviolet-visible spectrum and fluorescence spectrum of copper nanoclusters;
fig. 4 is a TEM image of copper nanoclusters: FIG. A is a TEM image, and the inset is a particle size distribution; FIG. 4B is a HRTEM diagram;
FIG. 5 is an SEM image of copper nanoclusters;
FIG. 6 is an XPS diagram of copper rice clusters;
fig. 7 is stability of copper nanoclusters: FIG. 7A is a graph showing the effect of storage time stability; FIG. 7B is a graph of ultraviolet radiation time effects; FIG. 7C is a salt effect influence diagram; FIG. 7D is H 2 O 2 Oxidative influence diagram; FIG. 7E is a graph of pH effects; FIG. 7F is a temperature influence diagram;
FIG. 8 is a response of copper cluster to tryptophan detection: FIG. 8A is a graph showing interactions of different metal cations with copper nanoclusters; FIG. 8B is a graph showing interactions of different anions with copper nanoclusters; FIG. 8C is a graph showing interactions of various amino acids and sugars with copper rice clusters;
FIG. 9 is a graph showing the sensitivity of copper nanocluster detection for tryptophan: FIG. 9A is a graph of fluorescence spectra of tryptophan and copper nanoclusters at different concentrations; FIG. 9B is a graph showing the fluorescence intensity of copper nanoclusters as a function of tryptophan concentration; FIG. 9C is a graph of the linear relationship between fluorescence intensity of copper nanoclusters and tryptophan concentration;
FIG. 10 is a response of copper nanoclusters-tryptophan to detection of mercury ions;
FIG. 11 is a graph showing the sensitivity of copper nanoclusters-tryptophan to detect mercury ions: FIG. 11A is a graph of fluorescence spectra of the effect of different concentrations of mercury ions on copper nanoclusters-tryptophan; FIG. 11B is a graph showing the relationship between the fluorescence intensity of copper nanoclusters and tryptophan as a function of the concentration of mercury ions; FIG. 11C is a graph of the linear relationship between the fluorescence intensity of copper nanoclusters and tryptophan and the concentration of mercury ions;
FIG. 12 is a fluorescence spectrum of the copper nanocluster solution prepared in comparative example 1;
FIG. 13 is a fluorescence spectrum of the copper nanocluster solution prepared in comparative example 2;
fig. 14 is a flow chart of the method of the present invention.
Detailed Description
The preferred embodiments of the present invention will be described below with reference to the accompanying drawings, it being understood that the preferred embodiments described herein are for illustration and explanation of the present invention only, and are not intended to limit the present invention.
The raw materials of the invention are as follows:
1. copper sulfate pentahydrate (CuSO) 4 ·5H 2 O, molecular weight 249.69) is produced by the industrial district of North in the Bao of the Ming street in the east of Tianjin.
2. Sodium humate (C) 9 H 8 Na 2 O 4 Molecular weight 226.14) is manufactured by Chengdu Ai Keda chemical company.
3. Ascorbic acid (C) 6 H 8 O 6 Molecular weight 176.124) is produced by Tianjin light-chemical industry research institute.
4. Sodium hydroxide (NaOH) and hydrochloric acid (HCl) are manufactured by tueshoku chemical engineering, inc.
5. The water used for the experiments was 18.2 M.OMEGA.ultrapure water.
Example 1:
as shown in the preparation method of fig. 14, step 1: 0.1998g of sodium Humate (HAN) is weighed and dissolved in 3.8mL of ultrapure water to obtain a protective agent sodium humate solution, copper ion solution (0.0333 g of copper sulfate pentahydrate is dissolved in 1.0mL of ultrapure water) is weighed, the two solutions are mixed and stirred, and the pH value of the mixed solution is regulated to be 10-11;
step 2: adding a reducing agent Ascorbic Acid (AA) solution (0.0666 g of ascorbic acid is dissolved in 1.0mL of ultrapure water) into the mixed solution obtained in the step 1, placing the mixed solution on a constant-temperature magnetic stirrer for reaction at 60 ℃ for 10min, standing the mixed solution after the reaction is finished, and cooling the mixed solution to room temperature;
step 3: centrifuging the cooling mixed solution obtained in the step 2 for 15min in a high-speed centrifuge (12000 rpm/min) to obtain a pale yellow sodium humate-copper nanocluster solution at the upper layer, and then storing the solution in a refrigerator at 4 ℃ in a dark place.
The prepared fluorescent copper nanocluster solution is light yellow in visible light, and generates weak cyan fluorescence under 365nm ultraviolet lamp irradiation, as shown in fig. 1.
Example 2:
by controlling the variable method, the reaction conditions and the raw material consumption for preparing the copper nanocluster are changed, the mass ratio of ascorbic acid to copper sulfate is designed to be (1:1, 2:1,3:1,4:1,5:1, 6:1), the mass ratio of sodium humate to copper sulfate is designed to be (1:1, 2:1,3:1,4:1,5:1,6:1, 7:1), the optimal reaction time is respectively (5 min,10min,15min,20min,30min,50min,90 min), the optimal reaction temperature is respectively (20 ℃,30 ℃,50 ℃,60 ℃,80 ℃), and the pH is adjusted to be (3.0,4.0,6.0,7.0,8.0, 10.0, 12.0), the influence of the consumption of HAN and AA, the reaction temperature, time and the pH on the sodium humate-copper nanocluster is judged, and the optimal reaction conditions and the consumption are determined.
As shown in FIG. 2A, the fluorescence intensity tends to increase first and decrease later, when m (CuSO 4 ) When m (AA) =1:2, the fluorescence value is the maximum, and the optimal ratio is obtained. Similarly, as can be seen from FIG. 2B, the fluorescence value is the maximum when m (HAN): m (AA) =6:1, and the ratio is the optimum ratio. As can be seen from FIG. 2C, the fluorescence value was maximized at a reaction time of 10 min. As can be seen from FIG. 2D, the fluorescence value was maximized at a reaction temperature of 60 ℃. FIG. 2E shows that the fluorescence value is maximal at pH 10.
Test characterization:
(1) Spectrum, morphology and composition of sodium humate-copper nanocluster
Whether sodium humate-copper nanoclusters (HAN-CuNCs) were successfully prepared:
the corresponding ultraviolet spectrum and fluorescence spectrum were measured in a quartz cuvette with 2.0mL of the prepared HAN-CuNCs solution, and the results are shown in FIG. 3. FIG. 3 shows UV absorption spectra of HAN-CuNCs at 209nm, 262nm and 370nm, indicating aromatic ring structure in copper nanoclusters. The fluorescence spectrum shows that under excitation of 370nm, an emission peak at 458nm appears, which indicates that the copper nanocluster has cyan fluorescence and can be used as a fluorescent material.
HAN-CuNCs morphology and size:
and (3) dripping the HAN-CuNCs solution on a copper mesh for sample preparation, and observing by using a transmission electron microscope after the liquid volatilizes. The particle size was also measured by placing the ultrasonic HAN-CuNCs liquid in a Markov particle sizer, the results of which are shown in FIG. 4. FIG. 4A is a transmission electron microscope analysis spectrum of the prepared HAN-CuNCs, from which it can be seen that the prepared HAN-CuNCs are uniformly dispersed and spherical, the inset is a particle size distribution diagram, the average size is about 3.21nm, and FIG. 4B is a HRTEM image of the prepared HAN-CuNCs, the lattice spacing is 0.206nm, and the prepared HAN-CuNCs are consistent with Cu (111).
HAN-CuNCs constituent elements:
the liquid sample obtained was freeze-dried to obtain a solid, which was characterized on a scanning electron microscope and an X-ray photoelectron spectroscopy analyzer, and the results are shown in fig. 5 and 6, respectively. FIG. 5 is an SEM image of HAN-CuNCs, which is composed of four elements, C, N, O and Cu. FIG. 6A is an XPS image of HAN-CuNCs, with results consistent with SEM.
(2) Stability of sodium humate-copper nanoclusters
200uL of HAN-CuNCs solution is respectively taken, and 1.8mL of BR (pH=6) buffer solution is added for dilution and volume fixing, wherein the BR buffer solution is phosphoric acid, acetic acid or boric acid triacid buffer solution. Its storage stability, salt tolerance, photobleaching, oxidation resistance, pH and temperature stability were subsequently investigated. From FIG. 7A, it was found that the fluorescence value of the copper nanoclusters does not change much with time under the protection of sodium humate, indicating that the stability of HAN-CuNCs is high. FIG. 7B shows that the fluorescence value of the copper nanoclusters does not change much with time as the ultraviolet irradiation time increases, indicating that the photo-bleaching resistance of HAN-CuNCs is strong. FIG. 7C shows that the fluorescence value of the copper nanoclusters does not change much over time as the salt solution concentration increases, indicating that HAN-CuNCs is salt tolerant. FIG. 7D shows the following H 2 O 2 The increase of the solution concentration causes the fluorescence value of the copper nanocluster to be little changed along with time, which proves that the HAN-CuNCs has strong oxidation resistance and high stability. FIG. 7E shows that the fluorescence value of HAN-CuNCs does not change much with increasing BR buffer pH at pH=1 to 14, indicating that the pH stability of HAN-CuNCs is good. FIG. 7F shows that the fluorescence value of HAN-CuNCs tends to decrease with increasing temperature, but does not change much, indicating that HAN-CuNCs have good temperature stability.
Example 3:
sodium humate-copper nanocluster tryptophan responsiveness test
Respectively transferring 200 mu L of sodium humate-copper nanoclustersSolution and 100. Mu.L of 0.01M metal cation (K) + ,Cs + ,Na + ,Mg 2+ ,Ca 2+ ,Sr 2+ ,Ba 2+ ,Zn 2+ ,Cd 2+ ,Cu 2+ ,Pb 2+ ,Ni 2+ ,Hg 2+ ,Co 2+ ,Mn 2+ ,Sn 2+ ,Fe 2+ ,Fe 3 + ,Al 3+ ,Bi 3+ ,Cr 3+ (II), (III) and (S) 2- ,HPO 4 2- ,H 2 PO 4 - ,PO 4 3- ,I - ,F - ,HSO 3 - ,Cl - ,Br - ,C 2 O 4 2- ,Cit 3- ) And amino acids (Gle, sue, fre, ile, his, cys, met, pro, tyr, val, glu, asn, arg, lys, phe, gly, gin, ala, ser, leu, thr, asp, GSH, trp) in 2.0mL EP tube, then adding BR (ph=6) buffer solution 1.7mL for dilution to volume; in addition, 200 μl of sodium humate-copper nanoclusters were removed and placed in a 2.0mL EP tube, and diluted with 1.8mL BR (ph=6) buffer solution to a constant volume as a control. All solutions were measured for fluorescence intensity at 370nm excitation wavelength and 458nm emission wavelength.
FIG. 8 records experimental results, which demonstrate that other metal cations, anions and amino acids do not have a significant effect on the fluorescence of HAN-CuNCs, except for the significant fluorescence enhancement of copper nanoclusters when tryptophan (Trp) is added, demonstrating the specific response of HAN-CuNCs to Trp.
Example 4:
sodium humate-copper nanocluster tryptophan sensitivity test
200 mu L of sodium humate-copper nanocluster solution and 100 mu L of tryptophan solution with different concentrations are respectively removed in an EP tube with 2.0mL, and then 1.7mL of BR (pH=6) buffer solution is added for dilution and volume fixing; in addition, 200 μl of sodium humate-copper nanoclusters were removed and placed in a 2.0mL EP tube, and diluted with 1.8mL BR (ph=6) buffer solution to a constant volume as a control. All solutions were measured for fluorescence intensity at 370nm excitation wavelength and 458nm emission wavelength.
FIG. 9A shows that HAN-CuNCs fluorescence intensity gradually increases with increasing tryptophan solution concentrationWhen the Trp solution concentration was increased to 200. Mu.M, the fluorescence increase of HAN-CuNCs tended to be gentle. The cyan fluorescent copper nanocluster prepared by the method can realize detection of Trp. FIG. 9B shows that the change in fluorescence intensity of HAN-CuNCs shows a good linear relationship with Trp concentration, expressed as F/F 0 ＝1.22838+0.00528C(R 2 = 0.9823), the linear range is 25-200 μm, the detection limit is 0.289 μm, which indicates that the prepared HAN-CuNCs have better response and sensitivity to Trp.
Example 5:
sodium humate-copper nanocluster pair Hg 2+ Responsiveness test
Respectively transferring 100 μL of sodium humate-copper nanocluster solution and 100 μL of 0.01M interference solution into 2.0mL EP tube, adding 1.7mL of BR (pH=6) buffer solution for dilution, standing for 24h, and continuously adding 100 μL of Hg with different concentrations 2+ A solution; in addition, 100. Mu.L of sodium humate-copper nanocluster solution and 100. Mu.L of tryptophan solution with a concentration of 0.01M were removed, placed in a 2.0mL EP tube, diluted with 1.8mL of BR (pH=6) buffer solution to a constant volume as a control, and after 24 hours of placement, the fluorescence intensities of all the solutions at 370nm excitation wavelength and 458nm emission wavelength were measured.
FIG. 10 records the results of the experiment, which demonstrate that in addition to Hg addition 2+ When the HAN-CuNCs-Trp has obvious fluorescence quenching, other substances have no obvious influence on the fluorescence of the HAN-CuNCs-Trp, and the fact that the HAN-CuNCs-Trp has a function on Hg is proved 2+ Has specific response.
Example 6:
sodium humate-copper nanocluster pair Hg 2+ Sensitivity test
Respectively transferring 100 μL of sodium humate-copper nanocluster solution and 100 μL of tryptophan solution with the concentration of 0.01M into an EP tube with the concentration of 2.0mL, standing for 24h, and continuously adding 100 μL of Hg with different concentrations 2+ Solution, then adding 1.7mL of BR (pH=6) buffer solution for dilution and volume fixation; in addition, 100. Mu.L of sodium humate-copper nanocluster solution and 100. Mu.L of tryptophan solution with a concentration of 0.01M were removed, placed in a 2.0mL EP tube, and diluted with 1.8mL BR (pH=6) buffer solution to a constant volume as a control. Measurement ofAll solutions showed fluorescence intensities at 370nm excitation wavelength and 458nm emission wavelength.
FIG. 11A shows results with Hg 2+ The fluorescence intensity of HAN-CuNCs-Trp gradually decreases when Hg concentration increases 2+ When the concentration of the solution was increased to 100. Mu.M, the fluorescence of HAN-CuNCs was almost completely quenched. The cyan fluorescent copper nanocluster prepared by the invention can realize Hg 2+ Is a continuous check of (1). FIG. 11B shows the change in fluorescence intensity of HAN-CuNCs-Trp with Hg 2+ The concentrations exhibit a good linear relationship, expressed as F/F 0 ＝0.9378+0.0023C(R 2 = 0.9953), the linear range was 25 to 200. Mu.M, the detection limit was 0.159. Mu.M, indicating that the prepared HAN-CuNCs were resistant to Hg 2+ Has better response and sensitivity.
Comparative example 1:
the same as in example 1, except that sodium humate protective agent is not added
The fluorescence intensity of the solution was measured, and the fluorescence spectrum was shown in fig. 12, and it was found that the copper nanocluster solution having a cyan fluorescence property could not be successfully prepared without the presence of the sodium humate protective agent, because the formed copper nanoclusters were easily oxidized in air to form copper oxide, thereby losing the fluorescence property.
Comparative example 2
The same as in example 1, except that ascorbic acid was not added as a reducing agent
The fluorescence intensity of the solution was measured, and the fluorescence spectrum is shown in fig. 13, and the copper ions could not be reduced to copper atoms without the presence of a reducing agent, so that the copper nanocluster solution having fluorescence properties could not be successfully prepared.
The foregoing has shown and described the basic principles, principal features and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and that the above embodiments and descriptions are merely illustrative of the principles of the present invention, and various changes and modifications may be made without departing from the spirit and scope of the invention, which is defined in the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (4)
1. A preparation method of sodium humate-copper nanoclusters is characterized by comprising the following steps: comprising the following steps:
step 1: weighing sodium humate, dissolving in ultrapure water to obtain a sodium humate solution as a protective agent, weighing copper salt, dissolving in ultrapure water to obtain a copper ion solution, mixing and stirring the two solutions, and regulating the pH value of the mixed solution to be 10-11;
step 2: adding an ascorbic acid solution serving as a reducing agent into the mixed solution obtained in the step 1, placing the mixed solution on a constant-temperature magnetic stirrer, reacting for 10min at 60 ℃, standing the mixed solution after the reaction is finished, and cooling to room temperature;
step 3: centrifuging the cooling mixed solution obtained in the step 2 in a high-speed centrifuge for 15min to obtain a pale yellow sodium humate-copper nanocluster solution at the upper layer, and then preserving the solution in a refrigerator at 4 ℃ in a dark place;
the copper ion solution in the step 1 is any one of copper nitrate, copper chloride, copper sulfate and copper acetate;
the mass ratio of the sodium humate to the ascorbic acid to the copper ions is 6:2:1;
the step 3 high speed centrifuge was run at 12000 rpm/min.
2. The use of sodium humate-copper nanoclusters obtained by the preparation method according to claim 1, characterized in that: the prepared sodium humate-copper nanocluster can generate cyan fluorescence, the maximum excitation wavelength is 370nm, and the maximum emission wavelength is 458nm, and is used for fluorescence enhancement type tryptophan detection.
3. The use of sodium humate-copper nanoclusters according to claim 2, characterized in that: for continuous fluorescence quenching type detection of mercury ions.
4. The use of sodium humate-copper nanoclusters as claimed in claim 3, characterized in that: the continuous measurement of mercury ions is realized by utilizing the sodium humate-copper nanocluster-tryptophan and the weakening change of the fluorescence intensity at the position of 458nm of the fluorescence emission spectrum.
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