CN115128051A - Fluorescence detection method for detecting 3-nitropropionic acid in sugarcane and sugarcane products - Google Patents
Fluorescence detection method for detecting 3-nitropropionic acid in sugarcane and sugarcane products Download PDFInfo
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- NWZSZGALRFJKBT-KNIFDHDWSA-N (2s)-2,6-diaminohexanoic acid;(2s)-2-hydroxybutanedioic acid Chemical compound OC(=O)[C@@H](O)CC(O)=O.NCCCC[C@H](N)C(O)=O NWZSZGALRFJKBT-KNIFDHDWSA-N 0.000 claims description 11
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- 238000004440 column chromatography Methods 0.000 claims description 6
- 239000012452 mother liquor Substances 0.000 claims description 6
- SMQUZDBALVYZAC-UHFFFAOYSA-N salicylaldehyde Chemical compound OC1=CC=CC=C1C=O SMQUZDBALVYZAC-UHFFFAOYSA-N 0.000 claims description 6
- 238000002189 fluorescence spectrum Methods 0.000 claims description 5
- 230000035484 reaction time Effects 0.000 claims description 5
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- 238000000746 purification Methods 0.000 claims description 2
- AIYIPTFRVFQOMC-UHFFFAOYSA-N pyren-1-ylmethylidenehydrazine Chemical compound C1=C2C(C=NN)=CC=C(C=C3)C2=C2C3=CC=CC2=C1 AIYIPTFRVFQOMC-UHFFFAOYSA-N 0.000 claims description 2
- RCYFOPUXRMOLQM-UHFFFAOYSA-N pyrene-1-carbaldehyde Chemical compound C1=C2C(C=O)=CC=C(C=C3)C2=C2C3=CC=CC2=C1 RCYFOPUXRMOLQM-UHFFFAOYSA-N 0.000 claims description 2
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- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
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- YISPKQZWSIMXMX-UHFFFAOYSA-N 3-nitropropan-1-ol Chemical class OCCC[N+]([O-])=O YISPKQZWSIMXMX-UHFFFAOYSA-N 0.000 description 1
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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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- 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
- G01N2021/6417—Spectrofluorimetric devices
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- G—PHYSICS
- G01—MEASURING; TESTING
- 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
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Abstract
The invention discloses a fluorescence detection method for detecting 3-nitropropionic acid in sugarcane and sugarcane products, which comprises the following steps: (1) preparing a pyrene derivative fluorescence sensor; (2) preparing a standard solution; (3) drawing a standard curve; the fluorescence value of the fluorescence intensity at 456nm and the 3-nitropropionic acid standard solution with the concentration within the range of 0.5-10.0 mu mol/L form a good linear relation, and a linear regression equation is obtained: f 456nm 1040.57+ 33.13C; the 3-nitropropionic acid standard solution with the absorbance at the fluorescence value of 456nm within the range of 10.0-100.0 mu mol/L has good linear relation, and the obtained linear regression equation: f 456nm 1325.93+ 9.39C. The process for simultaneously detecting the 3-nitropropionic acid by using the fluorescent sensor for detecting the 3-nitropropionic acid has the advantages of simple operation, quick response, high sensitivity, good selectivity and low cost.
Description
Technical Field
The invention belongs to the technical field of electrochemical detection methods, and particularly relates to a fluorescence detection method for detecting 3-nitropropionic acid in sugarcane and sugarcane products.
Background
3-Nitropropionic acid is a highly toxic neurotoxin, a metabolite of 3-nitropropanol produced by a variety of fungi, widely distributed in moldy sugarcane. The good stability of the 3-nitropropionic acid can enable the 3-nitropropionic acid to be stored in the subsequent sugar refining process and remain in the sucrose, and the sucrose is one of the most common food additives in daily life and poses great threat to human health. Therefore, the method for sensitively and rapidly determining the content of the 3-nitropropionic acid, which is simple and convenient to operate and low in cost, has important practical significance.
The 3-nitropropionic acid detection method reported in the current literature is mainly chromatography, wherein a high performance liquid chromatography and high performance liquid chromatography-mass spectrometry combined method is the most common, and is also the detection method used by the current national standard, and the sensitivity and the accuracy are higher. However, the chromatograph is high in purchase and use cost, complex to operate, high in technical requirements for use and maintenance, difficult to realize intelligent, low-cost and rapid detection, and not very popular in agricultural product production bases and small and medium-sized enterprises.
Disclosure of Invention
The invention discloses a fluorescence detection method for detecting 3-nitropropionic acid in sugarcane and sugarcane products, aiming at the technical problems that the method for detecting 3-nitropropionic acid in the prior art needs high-cost professional technicians and fixed detection places, almost all needs to carry out complex pretreatment processes, is complex to operate and time-consuming, and does not have advantages in rapid screening of samples.
In order to achieve the purpose, the technical scheme provided by the invention is as follows:
a fluorescence detection method for detecting 3-nitropropionic acid in sugarcane and sugarcane products comprises the following operation steps:
(1) preparation of pyrene derivative fluorescence sensor: dissolving 1-pyrene formaldehyde in tetrahydrofuran solution, stirring at room temperature to completely dissolve the 1-pyrene formaldehyde, adding hydrazine hydrate, stirring at room temperature, carrying out vacuum filtration on a product obtained by the reaction, washing with diethyl ether, and drying to obtain pyrene formaldehyde hydrazone; dissolving the obtained pyrene formaldehyde hydrazone and 4-tert-butyl-2, 6-formylphenol in tetrahydrofuran, dropwise adding glacial acetic acid, reacting at normal temperature for 24 hours, filtering, concentrating the filtrate, performing separation and purification by using a column chromatography method, standing to volatilize a solvent, and finally obtaining a pyrene derivative solid; dissolving the pyrene derivative solid in dimethyl sulfoxide to prepare a storage solution of the pyrene derivative fluorescent sensor;
(2) preparation of a standard solution: taking water as a solvent, dissolving 3-nitropropionic acid solid in the water to prepare mother liquor, and respectively mixing and diluting a certain amount of the mother liquor to obtain a series of mixed standard solutions to be detected with different concentrations, namely 3-nitropropionic acid standard solutions;
(3) drawing a standard curve: mixing the storage liquid of the pyrene derivative fluorescence sensor obtained in the step (1) and the 3-nitropropionic acid standard solution obtained in the step (2) according to a certain proportion, standing for a certain time, performing fluorescence emission spectrum scanning within the range of absorption wavelength of 375-700 nm, recording the spectrum, drawing a standard curve by using the fluorescence intensity value measured at 456nm, and calculating to obtain a linear regression equation; in the process of actually detecting the 3-nitropropionic acid, replacing the 3-nitropropionic acid standard solution with a sample to be detected, and respectively substituting the fluorescence values measured at 456nm into the obtained linear regression equation according to the same operation to calculate the content of the 3-nitropropionic acid in the sample to be detected;
the fluorescence value of the fluorescence intensity at 456nm and the 3-nitropropionic acid standard solution with the concentration within the range of 0.5-10.0 mu mol/L form a good linear relation, and the obtained linear regression equation is as follows: f 456nm 1040.57+33.13C, phaseCoefficient of correlation R is 0.9930, where F 456nm The fluorescence intensity value at 456nm, C is the concentration of 3-nitropropionic acid;
the 3-nitropropionic acid standard solution with the fluorescence value of 456nm and the absorbance within the range of 10.0-100.0 mu mol/L has good linear relation, and the obtained linear regression equation is as follows: f 456nm 1325.93+9.39C, correlation coefficient R is 0.9904; the detection limit of the method is as follows: 0.32. mu. mol/L.
Preferably, the amount of 1-pyrenecarboxaldehyde in the step (1) is 0.5756g (2.5mmol), and the amount of tetrahydrofuran is 10 mL.
Preferably, the hydrazine hydrate in the step (1) has a volume percentage of 25 percent, the using amount is 4.85mL (25mmol), and the quantity ratio of the substances of 1-pyrene formaldehyde and hydrazine hydrate is 1: 10; the reaction time of the 1-pyrene formaldehyde and hydrazine hydrate is 12 hours.
Preferably, the substance quantity ratio relation of pyrene formaldehyde hydrazone and 4-tertiary butyl-2, 6-formoxyl phenol in the step (1) is 1: 1.1; the reaction time of pyrenecarboxaldehyde hydrazone and 4-tert-butyl-2, 6-formylphenol was 24 hours.
Preferably, the eluent for the column chromatography in the step (1) is petroleum ether and ethyl acetate, and the volume ratio is 16:1, 12:1, 10:1, 8:1, 6:1 and 4:1 in sequence.
Preferably, a stock solution of the pyrene derivative fluorescence sensor is prepared at a concentration of 15. mu. mol/L in step (1).
Preferably, the volume ratio of the dimethyl sulfoxide solvent to the water in the step (1) is 2: 1.
Preferably, the standing time in step (3) is 30 min.
Compared with the prior art, the invention has the following beneficial effects:
the fluorescent sensor for detecting 3-nitropropionic acid is a pyrene derivative, the preparation process is simple, and the process for detecting 3-nitropropionic acid has the advantages of simplicity in operation, high response speed, high sensitivity, good selectivity and low cost.
Drawings
FIG. 1 is a mass spectrum of the pyrene derivative biosolids produced in step (1) of the present invention.
FIG. 2 is a graph of a fluorescence spectrum and a standard curve of the method of the present invention; wherein a is a fluorescence spectrogram of 3-nitropropionic acid with different concentrations in pyrene derivative fluorescence sensor solution; b is a standard curve of the fluorescence intensity of 3-nitropropionic acid at 456nm at different concentrations and the corresponding concentrations.
Detailed Description
The following detailed description is to be read in connection with the accompanying drawings, but it is to be understood that the scope of the invention is not limited to the specific embodiments. The raw materials and reagents used in the examples were all commercially available unless otherwise specified.
Example 1
A fluorescence detection method for detecting 3-nitropropionic acid in sugarcane and sugarcane products comprises the following specific operation steps:
(1) preparation of pyrene derivative fluorescence sensor: 0.5756g (2.5mmol) of 1-pyrene formaldehyde is dissolved in 10mL of tetrahydrofuran solution, the mixture is stirred at room temperature to be completely dissolved, 25% of hydrazine hydrate by mass percent is added, the dosage is 4.85mL (25mmol), the quantity ratio of the 1-pyrene formaldehyde to the hydrazine hydrate is 1:10, the mixture is stirred at room temperature for reaction for 12 hours, the product obtained by the reaction is subjected to reduced pressure suction filtration and washed by ether, and the product is dried to obtain pyrene formaldehyde hydrazone; 0.2441g (1.0mmol) of pyrene formaldehyde hydrazone and 0.2268g (1.1mmol) of 4-tert-butyl-2, 6-formylphenol are dissolved in 10mL of tetrahydrofuran, 100 mu L of glacial acetic acid is dropwise added to react for 24 hours at normal temperature, the mixture is filtered, filtrate is concentrated and is separated and purified by column chromatography, eluent of the column chromatography is petroleum ether and ethyl acetate, the volume ratio of the petroleum ether to the ethyl acetate is 16:1, 12:1, 10:1, 8:1, 6:1 and 4:1 in sequence, the solvent is volatilized after the mixture is placed, finally, pyrene derivative solid is obtained, and the pyrene derivative obtained by preparation is subjected to mass spectrometry, and the result is shown in figure 1; 0.0324g of pyrene derivative solid was dissolved in 50mL of dimethyl sulfoxide to obtain 1.5X 10 -3 1.5mL of pyrene derivative solution (1.5X 10) was added to the mol/L pyrene derivative solution -3 mol/L) gradually adding dimethyl sulfoxide to dilute to 100mL, and preparing a storage solution of the pyrene derivative fluorescence sensor with the concentration of 15 mu mol/L; the volume ratio of the dimethyl sulfoxide solvent to the water is 2: 1;
(2) preparation of a standard solution: taking water as solvent, taking 3-nitropropaneThe acid solid is dissolved in water to obtain a solution with a concentration of 3.0 × 10 -4 Respectively taking a certain amount of mother liquor for mixing and diluting the mother liquor of mol/L, and performing constant volume to obtain a series of 3-nitropropionic acid mixed standard solutions to be tested, namely 3-nitropropionic acid standard solutions, with the concentrations of 1.5, 3.0, 6.0, 9.0, 12.0, 24.0, 30.0, 60.0, 90.0, 120.0, 180.0, 240.0 and 300.0 mu mol/L;
(3) drawing a standard curve: using a fluorescence spectrophotometer model 960MC, the spectral scan parameters were set as follows: the measurement mode is wavelength scanning, the scanning mode is an emission mode, the excitation wavelength is 360nm, the scanning speed is 1000nm/min, the scanning interval is 1nm, and the excitation bandwidth and the emission bandwidth are 5 nm; mixing the storage liquid (15 mu mol/L) of the pyrene derivative fluorescence sensor obtained in the step (1) with the 3-nitropropionic acid standard solution obtained in the step (2) according to the volume ratio of 2:1, standing for 30min, carrying out fluorescence emission spectrum scanning within the range of absorption wavelength of 375-700 nm, recording a spectrum, drawing a standard curve by using a fluorescence intensity value measured at 456nm, and calculating to obtain a linear regression equation, wherein the concentration of the pyrene derivative fluorescence sensor is 10 mu mol/L, and the concentrations of the 3-nitropropionic acid are respectively 0.5, 1.0, 2.0, 4.0, 8.0, 10.0, 20.0, 40.0, 60.0, 80.0 and 100.0 mu mol/L;
the fluorescence intensity of the fluorescence value at 456nm and the 3-nitropropionic acid standard solution with the concentration within the range of 0.5-10.0 mu mol/L form a good linear relation, and the obtained linear regression equation is as follows: f 456nm 1040.57+33.13C, correlation coefficient R0.9930, wherein F456nm is the fluorescence intensity value at 456nm, C is the concentration of 3-nitropropionic acid; substituting the measured fluorescence values into the linear equation of FIG. 2 (b);
the 3-nitropropionic acid standard solution with the fluorescence value of 456nm and the absorbance within the range of 10.0-100.0 mu mol/L has good linear relation, and the obtained linear regression equation is as follows: f 456nm 1325.93+9.39C, correlation coefficient R is 0.9904; the detection limit of the method is as follows: 0.32 mu mol/L;
in the process of actually detecting the 3-nitropropionic acid, taking 20g of a sample of the peeled sugar cane, cutting the sample into small pieces, crushing the small pieces, uniformly mixing the small pieces to prepare a sample, and storing the sample at the temperature of minus 20 ℃ for later use; weighing 2g of a sample, adding 10mL of deionized water, carrying out ultrasonic extraction for 10min, centrifuging at 10000r/min for 5min, transferring the supernatant into another centrifuge tube, repeatedly extracting with 10mL of deionized water for 2 times, and combining the supernatants to obtain a sample to be detected; replacing the 3-nitropropionic acid standard solution in the step (3) with a sample to be detected, respectively substituting the fluorescence values measured at 456nm into the obtained linear regression equation according to the same operation, calculating the content of the 3-nitropropionic acid in the sample to be detected, calculating the corresponding 3-nitropropionic acid concentration value from the standard curve according to the measured fluorescence intensity, and calculating the standard recovery rate, wherein the result is shown in table 1:
TABLE 1 measurement results of 3-nitropropionic acid standard recovery in sugar cane samples
| Sample content (μmol/L) | Scalar quantity (mu mol/L) | Measured value (μmol/L) | Recovery (%) |
| 0.0 | 1.00 | 1.14 | 114.0 |
| 0.0 | 10.00 | 10.63 | 106.3 |
| 0.0 | 40.00 | 44.31 | 110.8 |
As can be seen from Table 1, the content of the 3-nitropropionic acid in the sugarcane sample is 0, and the fluorescent detection method for the 3-nitropropionic acid in the sugarcane sample provided by the invention has good practicability and accuracy.
FIG. 1 is a mass spectrum of the pyrene derivative obtained by the present invention, and as shown in FIG. 1, a proton peak appeared at m/z-433.19, which corresponds to a theoretical value of m/z-433.18, indicating that the pyrene derivative was successfully synthesized in step (1).
FIG. 2a is a fluorescence spectrum of 3-nitropropionic acid with different concentrations in a pyrene derivative fluorescence sensor solution, and as shown in the figure, the fluorescence emission peak at 456nm is gradually increased along with the increase of the concentration of the 3-nitropropionic acid; b is a calibration curve of the fluorescence intensity of the 3-nitropropionic acid with different concentrations at 456nm and the corresponding concentration, as shown in the figure, the fluorescence intensity of the fluorescence value at 456nm and the 3-nitropropionic acid standard solution with the concentration in the range of 0.5-10.0 mu mol/L form a good linear relation, and the obtained linear regression equation is as follows: f 456nm 1040.57+ 33.13C; the 3-nitropropionic acid standard solution with the absorbance at 456nm of the fluorescence value within the range of 10.0-100.0 mu mol/L has a good linear relation, and the obtained linear regression equation is as follows: f 456nm ＝1325.93+9.39C。
The fluorescence detection method has the characteristics of simplicity in operation, low cost, quick response, short reaction time, good reproducibility and the like, and can become a reliable means for low-cost and quick detection of the 3-nitropropionic acid.
The foregoing descriptions of specific exemplary embodiments of the present invention have been presented for purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain certain principles of the invention and its practical application to enable one skilled in the art to make and use various exemplary embodiments of the invention and various alternatives and modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims and their equivalents.
Claims (8)
1. A fluorescence detection method for detecting 3-nitropropionic acid in sugarcane and sugarcane products is characterized by comprising the following operation steps:
(1) preparation of pyrene derivative fluorescence sensor: dissolving 1-pyrene formaldehyde in a tetrahydrofuran solution, stirring to dissolve the 1-pyrene formaldehyde, adding hydrazine hydrate, stirring, carrying out suction filtration on a product obtained by the reaction, cleaning, and drying to obtain pyrene formaldehyde hydrazone; dissolving the obtained pyrene formaldehyde hydrazone and 4-tert-butyl-2, 6-formylphenol in tetrahydrofuran, dropwise adding glacial acetic acid, reacting at normal temperature, filtering, concentrating the filtrate, performing separation and purification by using a column chromatography method, standing to volatilize the solvent, and finally obtaining a pyrene derivative solid; dissolving a pyrene derivative solid in dimethyl sulfoxide to prepare a storage liquid of the pyrene derivative fluorescence sensor;
(2) preparation of a standard solution: taking water as a solvent, dissolving 3-nitropropionic acid solid in the water to prepare mother liquor, and respectively mixing and diluting a certain amount of the mother liquor to obtain a series of mixed standard solutions to be detected with different concentrations, namely 3-nitropropionic acid standard solutions;
(3) drawing a standard curve: mixing the storage liquid of the pyrene derivative fluorescence sensor obtained in the step (1) and the 3-nitropropionic acid standard solution obtained in the step (2) according to a certain proportion, standing for a certain time, performing fluorescence emission spectrum scanning within the range of absorption wavelength of 375-700 nm, recording a spectrum, drawing a standard curve by using a fluorescence intensity value measured at 456nm, and calculating to obtain a linear regression equation; in the process of actually detecting the 3-nitropropionic acid, replacing the 3-nitropropionic acid standard solution with a sample to be detected, and respectively substituting the fluorescence values measured at 456nm into the obtained linear regression equation according to the same operation to calculate the content of the 3-nitropropionic acid in the sample to be detected;
the fluorescence value of the fluorescence intensity at 456nm and the 3-nitropropionic acid standard solution with the concentration within the range of 0.5-10.0 mu mol/L form a good linear relation, and the obtained linear regression equation is as follows: f 456nm 1040.57+33.13C, correlation coefficient R0.9930, where F 456nm Is the fluorescence intensity value at 456nmC is the concentration of 3-nitropropionic acid;
the 3-nitropropionic acid standard solution with the fluorescence value of 456nm and the absorbance within the range of 10.0-100.0 mu mol/L has good linear relation, and the obtained linear regression equation is as follows: f 456nm 1325.93+9.39C, correlation coefficient R is 0.9904; the detection limit of the method is as follows: 0.32. mu. mol/L.
2. The fluorescence detection method for detecting 3-nitropropionic acid in sugarcane and sugarcane products according to claim 1, wherein the fluorescence detection method comprises the following steps: the amount of 1-pyrene carboxaldehyde in the step (1) is 0.5756g (2.5mmol) and the amount of tetrahydrofuran is 10 mL.
3. The fluorescence detection method for detecting 3-nitropropionic acid in sugarcane and sugarcane products according to claim 1, wherein the fluorescence detection method comprises the following steps: the volume percentage of the hydrazine hydrate in the step (1) is 25%, the using amount of the hydrazine hydrate is 4.85mL, and the quantity ratio relation of the 1-pyrene formaldehyde to the hydrazine hydrate is 1: 10; the reaction time of the 1-pyrene formaldehyde and hydrazine hydrate is 12 hours.
4. The fluorescence detection method for detecting 3-nitropropionic acid in sugarcane and sugarcane products according to claim 1, wherein the fluorescence detection method comprises the following steps: in the step (1), the quantitative ratio relation between pyrene formaldehyde hydrazone and 4-tert-butyl-2, 6-formylphenol is 1: 1.1; the reaction time of pyrenecarboxaldehyde hydrazone and 4-tert-butyl-2, 6-formylphenol was 24 hours.
5. The fluorescence detection method for detecting 3-nitropropionic acid in sugarcane and sugarcane products according to claim 1, wherein the fluorescence detection method comprises the following steps: the eluent of the column chromatography in the step (1) is petroleum ether and ethyl acetate, and the volume ratio of the eluent is 16:1, 12:1, 10:1, 8:1, 6:1 and 4:1 in sequence.
6. The fluorescence detection method for detecting 3-nitropropionic acid in sugarcane and sugarcane products according to claim 1, wherein the fluorescence detection method comprises the following steps: and (2) preparing a storage solution of the pyrene derivative fluorescence sensor with the concentration of 15 mu mol/L in the step (1).
7. The fluorescence detection method for detecting 3-nitropropionic acid in sugarcane and sugarcane products according to claim 1, wherein the fluorescence detection method comprises the following steps: in the step (1), the volume ratio of the dimethyl sulfoxide solvent to the water is 2: 1.
8. The fluorescence detection method for detecting 3-nitropropionic acid in sugarcane and sugarcane products according to claim 1, wherein the fluorescence detection method comprises the following steps: the standing time in the step (3) is 30 min.
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