CN114813698A - Method for detecting phosphate in aquaculture water based on surface enhanced Raman spectroscopy - Google Patents
Method for detecting phosphate in aquaculture water based on surface enhanced Raman spectroscopy Download PDFInfo
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- 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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Abstract
The invention provides a method for detecting phosphate in aquaculture water based on surface enhanced Raman spectroscopy, which is characterized in that a nano silver solution is used as an active substrate, rhodamine 6G is used as a probe, and a coagulant is added, so that a Raman signal can be enhanced, and accurate detection of trace phosphate in aquaculture water is realized. The detection method provided by the invention is slightly influenced by the aquaculture water environment, has certain universality, has the characteristics of higher precision, higher sensitivity and higher stability compared with the existing method, is simple and convenient to operate, has easily available reagents, can be matched with portable Raman spectrum detection equipment to carry out field detection, and is convenient and rapid.
Description
Technical Field
The invention belongs to the technical field of phosphate analysis and detection, and particularly relates to a method for detecting phosphate in aquaculture water based on surface enhanced Raman spectroscopy.
Background
In recent years, a large amount of phosphate is released into an aqueous environment by means of industrial manufacturing, agricultural production, municipal sewage, and the like, resulting in the presence of an excessive amount of phosphate in the water body. And excessive phosphate can stimulate mass propagation of algae and bacteria, so that water eutrophication is caused, and fishes die in mass. At present, water eutrophication has become one of the major threats to the sustainable development of the aquaculture industry. Therefore, the method can quickly and accurately detect the content of phosphate in the aquaculture water and prevent water eutrophication, and is very necessary and urgent for the development of fishery production.
At present, various detection methods such as spectrophotometry, fluorescence measurement, phosphorescence spectrometry, electrochemistry, colorimetry, ion chromatography, biosensor method and the like are available for detecting phosphate in different water environments. The most widely used ammonium molybdate spectrophotometry has excellent stability, but the detection limit is usually above 0.01mg/L, and a complicated pretreatment procedure is required. Ion chromatography and fluorescence spectroscopy have the advantage of being fast and accurate, but require high precision equipment to assist in detection. Electrochemical sensors are small, highly accurate, and ion selective, but require complex calibration before use and often have a short lifetime. The Surface Enhanced Raman Spectroscopy (SERS) technology has the characteristics of high sensitivity and strong stability, and can be used for trace and even single molecule detection. However, the research of detecting phosphate in aquaculture by using the surface enhanced raman spectroscopy technology has not been reported at present due to the reasons of more interference factors (various interfering ions, temperature, dissolved oxygen, PH, and the like), interconversion between phosphorus-containing substances, weak raman signal of phosphate in water and difficult adsorption on the surface of an enhanced substrate.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a method for detecting phosphate in aquaculture water based on surface enhanced Raman spectroscopy, which is established by taking nano-silver as an enhanced substrate and rhodamine 6G as a probe and can sensitively determine the phosphate in the aquaculture water.
The present invention achieves the above-described object by the following technical means.
A method for detecting phosphate in aquaculture water based on surface enhanced Raman spectroscopy comprises the following steps:
step 1: preparation of a standard test system for phosphate at known concentrations: sequentially adding 6-12 mu L10 into a test tube - 4 10-30 μ L of 2 × 10 mol/L R6 aqueous solution of 6G -2 15-30 mu L of 6mol/L H mol/L ammonium molybdate aqueous solution 2 SO 4 Adding 200 mu L of solution to be detected, then adding deionized water to dilute to 0.3ml, uniformly mixing, and reacting for 10 minutes at room temperature; then sequentially adding 1.5ml of nano silver solution and 50-100 mu L of 1mol/L NaCl aqueous solution into the test tube, diluting the solution to 2ml by using distilled water, and continuing to react for 10 minutes at room temperature;
step 2: preparing a blank control system without adding the solution to be detected according to the step 1;
and step 3: setting instrument parameters of a Raman spectrometer, respectively placing a standard test system and a blank comparison system in a quartz cuvette, and scanning by the Raman spectrometer to obtain a Raman spectrum curve;
and 4, step 4: 1508cm standard test system for measurement -1 The intensity value of the Raman characteristic peak is measured, and the 1508cm of the blank control system is measured at the same time -1 The intensity value of the Raman characteristic peak is calculated, and the 1508cm of the standard test system relative to the blank control system is calculated -1 The variation of the intensity of the Raman characteristic peak; and then comparing the intensity variation of the Raman characteristic peak with a Raman spectrum working curve of the phosphate to obtain the concentration of the phosphate.
Further, the preparation method of the nano silver solution in the step 1 comprises the following steps:
adding 100-120 mu L of silver nitrate water solution with the concentration of 0.1mol/L and 80-100 mg of sodium citrate into 100-150 ml of deionized water, dropwise adding 100-150 mu L of sodium borohydride water solution with the concentration of 0.01mol/L under magnetic stirring, continuously stirring for 5 minutes, carrying out water bath until the liquid becomes bright yellow, and cooling at room temperature to obtain nano silver solution serving as an enhancing reagent.
Further, in the preparation process of the nano-silver solution, the temperature of the water bath is 70-90 ℃, and the stirring is continuously carried out for 6-9 hours in the water bath.
Further, the nano silver solution is refrigerated and stored at 4 ℃ in the dark after being prepared.
Further, the preparation method of the solution to be tested in the step 1 comprises the following steps:
firstly, accurately weighing 1.6394g of sodium phosphate, and fixing the volume to 100ml by using deionized water to obtain 0.1mol/L phosphate standard solution; and then diluting the phosphate standard solution to different concentrations in a gradient manner by using deionized water, so as to obtain a standard working solution of phosphate, namely the solution to be detected.
Further, in the step 4, the standard test system 1508cm -1 Has a Raman characteristic peak intensity value of I and a blank control system of 1508cm -1 Has a Raman characteristic peak intensity value of I 0 The standard test system is 1508cm in comparison with the blank control system -1 The variation of Raman characteristic peak intensity is delta I, and delta I is I 0 -I；
Comparing the Raman characteristic peak intensity variation with a Raman spectrum working curve of phosphate to obtain a linear regression equation:
wherein C represents a phosphate concentration.
Further, in step 3, the laser power of the raman spectrometer is set to 150mW, and the exposure time for data collection is set to 5 s.
The invention has the following beneficial effects:
according to the invention, the nano-silver solution is used as an active substrate, the rhodamine 6G is used as a probe, and the coagulant is added, so that the Raman signal can be effectively enhanced, and the accurate detection of trace phosphate in the aquaculture water can be realized. In addition, the detection method provided by the invention is slightly influenced by the aquaculture water environment, has certain universality, has the characteristics of higher precision, higher sensitivity and higher stability compared with the existing method, is simple and convenient to operate, has easily available reagents, can be matched with portable Raman spectrum detection equipment to carry out field detection, and is convenient and rapid.
Drawings
FIG. 1 is a TEM image of nano-silver particles in the nano-silver solution described in example 1;
FIG. 2 is a TEM image of silver nanoparticles in the test system described in example 1;
FIG. 3 is a surface enhanced Raman spectrum of the detection method described in example 1 for various concentrations of phosphate standard solutions;
FIG. 4 is a plot of Δ I versus phosphate concentration for a standard working solution as described in example 1.
Detailed Description
The invention will be further described with reference to the following figures and specific examples, but the scope of the invention is not limited thereto.
Example 1:
the invention discloses a method for detecting phosphate in aquaculture water based on surface enhanced Raman spectroscopy, which comprises the following steps:
step 1: preparing a nano silver solution;
adding 118 mu L of silver nitrate aqueous solution with the concentration of 0.1mol/L and 88mg of sodium citrate into 120ml of deionized water; adding 100 mu L of sodium borohydride aqueous solution with the concentration of 0.01mol/L dropwise under magnetic stirring, continuing stirring for 5 minutes, then carrying out water bath to 80 ℃ and continuing stirring for 8 hours until the liquid becomes bright yellow, cooling at room temperature to obtain nano silver solution serving as an enhancing reagent, and then refrigerating at 4 ℃ and storing in the dark.
Step 2: 1.6394g of sodium phosphate is accurately weighed and is made to be 100ml by deionized water, thus obtaining 0.1mol/L phosphate standard solution.
And step 3: and (3) diluting the phosphate standard solution prepared in the step (2) to 0.2 mu mol/L, 0.4 mu mol/L, 0.8 mu mol/L, 2 mu mol/L, 4 mu mol/L, 8 mu mol/L, 12 mu mol/L, 16 mu mol/L and 20 mu mol/L by using deionized water in a gradient manner to obtain a standard working solution of phosphate, namely the solution to be detected.
And 4, step 4: preparing a phosphate standard test system with a known concentration;
10 μ L of 10 was added to a 5ml test tube in sequence -4 mol/L R6G aqueous solution, 20. mu.L 2X 10 -2 Ammonium molybdate aqueous solution of 20. mu.L and 6mol/L H 2 SO 4 200 mu L of the phosphate standard working solution prepared in the step 3, then adding deionized water to dilute to 0.3ml, uniformly mixing, and reacting at room temperature for 10 minutes; then, 1.5ml of the nano-silver solution prepared in step 1 and 50. mu.L of a 1mol/L NaCl aqueous solution were sequentially added to the test tube, and diluted to 2ml with deionized water, and the reaction was continued at room temperature for 10 minutes.
And 5: and (4) preparing a blank control system without adding a phosphate standard working solution according to the method in the step 4.
Step 6: respectively placing the phosphate standard test system prepared in step 4 and the blank control system prepared in step 5 in a quartz cuvette, and performing BWS465-785S (B)&W TEK) type Raman spectrometer, setting the laser power to be 150mW, setting the exposure time for data collection to be 5s, scanning to obtain the surface enhanced Raman spectrum curve of each system, and measuring to obtain a phosphate standard test system 1508cm -1 Has a Raman characteristic peak intensity value of I, and a blank control system 1508cm -1 Has a Raman characteristic peak intensity value of I 0 The standard phosphate test system is 1508cm in comparison with the blank control system -1 The variation of Raman characteristic peak intensity is delta I, and delta I is I 0 -I。
And 7: forming a Raman spectrum working curve of the phosphate by using the Raman spectrum curves of the standard phosphate working solutions with different concentrations, and making a working curve according to the concentration relation of the delta I to the phosphate calculated in the step 6, namely comparing the delta I with the Raman spectrum working curve of the phosphate to obtain a linear regression equation:
wherein, C represents phosphate concentration, the unit is μmol/L, and the linear range of the phosphate concentration in the phosphate standard test system determined in the embodiment is 0.2-20 μmol/L.
Fig. 1 and 2 are transmission electron microscope characterization (TEM) results of the nano silver solution and the testing system in this example, respectively; fig. 1 shows that the nano silver particles in the nano silver solution are in a better dispersed state, and fig. 2 shows that the nano silver particles in the test solution are in an aggregated state, which indicates that the nano silver particles provide a large number of raman hot spots in the test solution for the adhesion of R6G molecules, thereby playing a good role in enhancing.
Fig. 3 and 4 are a surface enhanced raman spectrum curve and a linear relationship curve of the detection method provided in this embodiment for different concentrations of phosphate standard solutions, respectively; the abscissa in fig. 3 represents the raman shift, and the ordinate represents the raman intensity.
Example 2:
the invention discloses a method for detecting phosphate in aquaculture water based on surface enhanced Raman spectroscopy, which comprises the following steps:
step 1: preparing a nano silver solution;
adding 100 mu L of silver nitrate aqueous solution with the concentration of 0.1mol/L and 80mg of sodium citrate into 100ml of deionized water; adding 120 mu L of sodium borohydride aqueous solution with the concentration of 0.01mol/L dropwise under magnetic stirring, continuing stirring for 5 minutes, then carrying out water bath to 70 ℃ and continuing stirring for 6 hours until the liquid becomes bright yellow, cooling at room temperature to obtain nano silver solution serving as an enhancing reagent, and then refrigerating at 4 ℃ and storing in the dark.
Step 2: 1.6394g of sodium phosphate is accurately weighed and is made to be 100ml by deionized water, thus obtaining 0.1mol/L phosphate standard solution.
And step 3: and (3) diluting the phosphate standard solution prepared in the step (2) to 0.2 mu mol/L, 0.4 mu mol/L, 0.8 mu mol/L, 2 mu mol/L, 4 mu mol/L, 8 mu mol/L, 12 mu mol/L, 16 mu mol/L and 20 mu mol/L by using deionized water in a gradient manner to obtain a standard working solution of phosphate, namely the solution to be detected.
And 4, step 4: preparing a phosphate standard test system with a known concentration;
add 6. mu.L 10 sequentially to 5ml test tube -4 mol/L R6G aqueous solution, 10. mu.L 2X 10 -2 mol/L ammonium molybdate aqueous solution, 15 μ L6 mol/L H 2 SO 4 200 mu L of the phosphate standard working solution prepared in the step 3, then adding deionized water to dilute to 0.3ml, uniformly mixing, and reacting at room temperature for 10 minutes; then, 1.5ml of the nano-silver solution prepared in step 1 and 80. mu.L of a 1mol/L NaCl aqueous solution were sequentially added to the test tube, and diluted to 2ml with deionized water, and the reaction was continued at room temperature for 10 minutes.
And 5: and (4) preparing a blank control system without adding a phosphate standard working solution according to the method in the step 4.
Step 6: respectively placing the phosphate standard test system prepared in the step 4 and the blank control system prepared in the step 5 in a quartz cuvette and performing detection in a BWS465-785S (B)&W TEK) type Raman spectrometer, setting the laser power to be 150mW, setting the exposure time for data collection to be 5s, scanning to obtain the surface enhanced Raman spectrum curve of each system, and measuring to obtain a phosphate standard test system 1508cm -1 Has a Raman characteristic peak intensity value of I, and a blank control system 1508cm -1 Has a Raman characteristic peak intensity value of I 0 Then the phosphate standard test system is 1508cm in length relative to the blank control system -1 The variation of Raman characteristic peak intensity is delta I, where 0 -I。
And 7: forming a Raman spectrum working curve of the phosphate by using the Raman spectrum curves of the standard phosphate working solutions with different concentrations, and making a working curve according to the concentration relation of the delta I to the phosphate calculated in the step 6, namely comparing the delta I with the Raman spectrum working curve of the phosphate to obtain a linear regression equation:
wherein C represents phosphate concentration in μmol/L.
Example 3:
the invention discloses a method for detecting phosphate in aquaculture water based on surface enhanced Raman spectroscopy, which comprises the following steps:
step 1: preparing a nano silver solution;
adding 120 mu L of silver nitrate aqueous solution with the concentration of 0.1mol/L and 100mg of sodium citrate into 150ml of deionized water; adding 150 mu L of sodium borohydride aqueous solution with the concentration of 0.01mol/L dropwise under magnetic stirring, continuing stirring for 5 minutes, then carrying out water bath to 90 ℃ and continuing stirring for 9 hours until the liquid becomes bright yellow, cooling at room temperature to obtain nano silver solution serving as an enhancing reagent, and then refrigerating at 4 ℃ and storing in the dark.
Step 2: 1.6394g of sodium phosphate is accurately weighed and is made to be 100ml by deionized water, thus obtaining 0.1mol/L phosphate standard solution.
And step 3: and (3) diluting the phosphate standard solution prepared in the step (2) to 0.2 mu mol/L, 0.4 mu mol/L, 0.8 mu mol/L, 2 mu mol/L, 4 mu mol/L, 8 mu mol/L, 12 mu mol/L, 16 mu mol/L and 20 mu mol/L by using deionized water in a gradient manner to obtain a standard working solution of phosphate, namely the solution to be detected.
And 4, step 4: preparing a phosphate standard test system with a known concentration;
adding 12 μ L10 in turn into 5ml test tube -4 mol/L R6G aqueous solution, 30. mu.L 2X 10 -2 mol/L ammonium molybdate aqueous solution, 30. mu.L 6mol/L H 2 SO 4 200 mu L of the phosphate standard working solution prepared in the step 3, then adding deionized water to dilute to 0.3ml, uniformly mixing, and reacting at room temperature for 10 minutes; then, 1.5ml of the nano silver solution prepared in step 1 and 100. mu.L of a 1mol/L NaCl aqueous solution were sequentially added to the test tube, and diluted to 2ml with deionized water, and the reaction was continued at room temperature for 10 minutes.
And 5: and (4) preparing a blank control system without adding a phosphate standard working solution according to the method in the step 4.
Step 6: respectively placing the phosphate standard test system prepared in the step 4 and the blank control system prepared in the step 5 in a quartz cuvette and performing detection in a BWS465-785S (B)&W TEK) type Raman spectrometer, setting the laser power to be 150mW, setting the exposure time for data collection to be 5s, scanning to obtain the surface enhanced Raman spectrum curve of each system, and measuring to obtain a phosphate standard test system 1508cm -1 Has a Raman characteristic peak intensity value of I, and a blank control system 1508cm -1 Has a Raman characteristic peak intensity value of I 0 The standard phosphate test system is 1508cm in comparison with the blank control system -1 The variation of Raman characteristic peak intensity is delta I, and delta I is I 0 -I。
And 7: forming a Raman spectrum working curve of the phosphate by the Raman spectrum curves of the standard phosphate working solutions with different concentrations, and working according to the concentration relation of the delta I to the phosphate calculated in the step 6, namely working the delta I and the Raman spectrum of the phosphateThe curves are compared to obtain a linear regression equation as follows:
wherein C represents phosphate concentration in μmol/L.
Example 4:
in this embodiment, the detection method provided in embodiment 1 is used for detecting a culture water sample, and specifically includes the following steps:
preparing four water samples including pond culture water, Yangjiang water, fish and vegetable symbiotic circulating water and tap water; then filtering the water sample by using a 0.45-micron microporous filter membrane, adding a phosphate standard solution into the four water samples by adopting a standard addition method, and preparing water samples to be detected containing phosphate with different concentrations;
obtaining a water sample to be tested 1508cm according to the method provided in the embodiment 2 -1 Intensity value I of Raman characteristic peak Sample (I) Calculating to obtain Delta I Sample (I) ＝I 0 -I Sample (I) And calculating the content of phosphate in the sample solution and the standard recovery rate by combining a phosphate concentration relation working curve, wherein the calculation result is shown in the following table 1, wherein RSD represents relative standard deviation:
table 1 example 4 water sample to be tested adding mark recovery test result
The present invention is not limited to the above-described embodiments, and any obvious improvements, substitutions or modifications can be made by those skilled in the art without departing from the spirit of the present invention.
Claims (7)
1. A method for detecting phosphate in aquaculture water based on surface enhanced Raman spectroscopy is characterized by comprising the following steps:
step 1: preparation of a standard test system for phosphate at known concentrations: sequentially adding 6-12 mu L10 into a test tube -4 10-30 μ L of 2 × 10 mol/L R6 aqueous solution of 6G -2 15-30 mu L of 6mol/L H mol/L ammonium molybdate aqueous solution 2 SO 4 Adding 200 mu L of solution to be detected, then adding deionized water to dilute to 0.3ml, uniformly mixing, and reacting at room temperature for 10 minutes; then sequentially adding 1.5ml of nano-silver solution and 50-100 mu L of 1mol/L NaCl aqueous solution into the test tube, diluting the solution to 2ml by using distilled water, and continuing to react for 10 minutes at room temperature;
and 2, step: preparing a blank control system without adding the solution to be detected according to the step 1;
and step 3: setting instrument parameters of a Raman spectrometer, respectively placing a standard test system and a blank comparison system in a quartz cuvette, and scanning by the Raman spectrometer to obtain a Raman spectrum curve;
and 4, step 4: 1508cm standard test system for measurement -1 The intensity value of the Raman characteristic peak is measured, and the 1508cm of the blank control system is measured at the same time -1 The intensity value of the Raman characteristic peak is calculated, and the 1508cm of the standard test system relative to the blank control system is calculated -1 The variation of the intensity of the Raman characteristic peak; and then comparing the intensity variation of the Raman characteristic peak with a Raman spectrum working curve of the phosphate to obtain the concentration of the phosphate.
2. The method for detecting phosphate in aquaculture water based on surface enhanced Raman spectroscopy according to claim 1, wherein the preparation method of the nano silver solution in the step 1 comprises the following steps:
adding 100-120 mu L of silver nitrate water solution with the concentration of 0.1mol/L and 80-100 mg of sodium citrate into 100-150 ml of deionized water, dropwise adding 100-150 mu L of sodium borohydride water solution with the concentration of 0.01mol/L under magnetic stirring, continuously stirring for 5 minutes, carrying out water bath until the liquid becomes bright yellow, and cooling at room temperature to obtain nano silver solution serving as an enhancing reagent.
3. The method for detecting phosphate in aquaculture water based on surface enhanced Raman spectroscopy according to claim 2, wherein in the preparation process of the nano-silver solution, the water bath temperature is 70-90 ℃, and the stirring is continuously carried out for 6-9 hours in the water bath.
4. The method for detecting phosphate in aquaculture water based on surface enhanced Raman spectroscopy of claim 2, wherein the nanosilver solution is refrigerated at 4 ℃ in the dark after being prepared.
5. The method for detecting phosphate in aquaculture water based on surface enhanced Raman spectroscopy according to claim 1, wherein the solution to be detected in step 1 is prepared by a method comprising:
firstly, accurately weighing 1.6394g of sodium phosphate, and fixing the volume to 100ml by using deionized water to obtain 0.1mol/L phosphate standard solution; and then diluting the phosphate standard solution to different concentrations in a gradient manner by using deionized water, so as to obtain a standard working solution of phosphate, namely the solution to be detected.
6. The method for detecting phosphate in aquaculture water based on surface enhanced Raman spectroscopy of claim 1, wherein in the step 4, the standard test system is 1508cm -1 Has a Raman characteristic peak intensity value of I and a blank control system of 1508cm -1 Has a Raman characteristic peak intensity value of I 0 The standard test system is 1508cm in comparison with the blank control system -1 The variation of Raman characteristic peak intensity is delta I, and delta I is I 0 -I；
Comparing the Raman characteristic peak intensity variation with a Raman spectrum working curve of phosphate to obtain a linear regression equation:
wherein C represents a phosphate concentration.
7. The method for detecting phosphate in aquaculture water based on surface enhanced Raman spectroscopy of claim 1, wherein in step 3, the laser power of the Raman spectrometer is set to 150mW, and the exposure time for data collection is set to 5 s.
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| CN116162277A (en) * | 2023-04-24 | 2023-05-26 | 北京市农林科学院智能装备技术研究中心 | Method for rapidly measuring phosphate radical in water |
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| US7989211B1 (en) * | 2006-09-27 | 2011-08-02 | Ut-Battelle, Llc | Functionalized gold surface-enhanced raman scattering substrate for rapid and ultra-sensitive detection of anionic species in the environment |
| CN106093002A (en) * | 2016-06-02 | 2016-11-09 | 中国原子能科学研究院 | Sulfate radical and the device and method of phosphate radical in a kind of high activity liquid waste of mensuration simultaneously |
| CN108459009A (en) * | 2018-05-07 | 2018-08-28 | 广西师范大学 | A method of measuring phosphate radical with Surface enhanced Raman spectroscopy |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| US7989211B1 (en) * | 2006-09-27 | 2011-08-02 | Ut-Battelle, Llc | Functionalized gold surface-enhanced raman scattering substrate for rapid and ultra-sensitive detection of anionic species in the environment |
| CN106093002A (en) * | 2016-06-02 | 2016-11-09 | 中国原子能科学研究院 | Sulfate radical and the device and method of phosphate radical in a kind of high activity liquid waste of mensuration simultaneously |
| CN108459009A (en) * | 2018-05-07 | 2018-08-28 | 广西师范大学 | A method of measuring phosphate radical with Surface enhanced Raman spectroscopy |
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| CN116162277A (en) * | 2023-04-24 | 2023-05-26 | 北京市农林科学院智能装备技术研究中心 | Method for rapidly measuring phosphate radical in water |
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