CN113533321A - Method for detecting nitrite by improving Griess reagent - Google Patents
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- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 title claims abstract description 71
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- PIICEJLVQHRZGT-UHFFFAOYSA-N Ethylenediamine Chemical compound NCCN PIICEJLVQHRZGT-UHFFFAOYSA-N 0.000 claims description 4
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- ZCUQOPGIJRGJDA-UHFFFAOYSA-N 1-naphthalen-1-ylethane-1,2-diamine Chemical compound C1=CC=C2C(C(N)CN)=CC=CC2=C1 ZCUQOPGIJRGJDA-UHFFFAOYSA-N 0.000 abstract description 5
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- 229910002651 NO3 Inorganic materials 0.000 abstract description 2
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 abstract description 2
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- 101001018064 Homo sapiens Lysosomal-trafficking regulator Proteins 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
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- SZVJSHCCFOBDDC-UHFFFAOYSA-N ferrosoferric oxide Chemical compound O=[Fe]O[Fe]O[Fe]=O SZVJSHCCFOBDDC-UHFFFAOYSA-N 0.000 description 2
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- 231100000315 carcinogenic Toxicity 0.000 description 1
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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
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
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- Life Sciences & Earth Sciences (AREA)
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Abstract
The invention relates to the technical field of chemical analysis and detection, in particular to a method for detecting nitrite by improving a Griess reagent. The method improves the existing Griess reagent for detecting nitrite, utilizes the catalytic oxidation characteristic of the copper-iodine doped carbon dot to accelerate the diazotization reaction of nitrite and sulfanilic acid and the coupling reaction of the nitrite and the naphthyl ethylenediamine hydrochloride, improves the detection sensitivity by 7-8 times, reduces the lowest nitrite detection limit from 0.06mg/kg to 0.01mg/kg in the method for detecting nitrite and nitrate in GB/T5009.33-2016 food safety national standard food, and is particularly suitable for the detection of nitrite with low content in blood, urine and food in vivo. The detection result of the method accords with the first method ion chromatography of GB 5009.33-2016. The method has the characteristics of high sensitivity, strong specificity, simple and rapid operation and the like.
Description
Technical Field
The invention relates to the technical field of chemical analysis and detection, in particular to a method for detecting nitrite by improving a Griess reagent.
Background
Nitric Oxide (NO) is an important signaling molecule in the human body and plays an irreplaceable regulatory role in the cardiovascular, nervous and immune systems, and NO is evaluated by Science as the "star molecule" in 1992 because of its important physiological functions. NO has the characteristics of simple structure, low molecular weight, lipophilicity, free radicals and the like, is extremely unstable in vivo, has a half-life period of only a few seconds, is easy to form nitrite, and reflects NO change through in vivo nitrite detection. Nitrite is also very important to detect in food, and nitrite can generate carcinogenic substances with related chemical substances, so that the nation is also paying more attention. At present, the detection method of nitrite mainly comprises ion chromatography and spectrophotometry. The national standard method for measuring nitrite in food is naphthyl ethylenediamine hydrochloride spectrophotometry, and the principle is that a sample is subjected to protein precipitation and fat removal, nitrite and sulfanilic acid are diazotized under the weak acid condition and then coupled with naphthyl ethylenediamine hydrochloride to form a mauve dye, and the mauve dye is compared with the standard and quantified by the spectrophotometry.
The nano enzyme is a mimic enzyme which has the unique performance of a nano material and also has a catalytic function, is a novel mimic enzyme, has the advantages which can not be achieved by other traditional mimic enzymes, can be researched and utilized by people according to the characteristics of the nano material mimic enzyme, and has a larger application prospect; the fact that ferroferric oxide has hydrogen peroxide nanometer mimic enzyme characteristics is proved, but direct application of ferroferric oxide is limited due to low enzyme activity.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a method for improving a Griess reagent to detect nitrite, which improves the existing method for detecting nitrite by using the Griess reagent, utilizes the catalytic oxidation characteristic of copper-iodine doped carbon dots to catalyze the diazotization reaction of nitrite and sulfanilic acid and the coupling reaction of the nitrite and naphthyl ethylenediamine hydrochloride, improves the detection sensitivity by 7-8 times, reduces the detection limit of nitrite from the lowest 0.06mg/kg in the method for detecting nitrite and nitrate in GB/T5009.33-2016 food safety national standard food to 0.01mg/kg, and is particularly suitable for the determination of low-content nitrite in blood, urine and food in vivo. The detection result of the method accords with the first method ion chromatography of GB 5009.33-2016. The method has the characteristics of high sensitivity, strong specificity, simple and rapid operation and the like.
The method for detecting nitrite by improving the Griess reagent comprises the following steps:
(1) making a nitrite working curve: adding 400 mu L of sulfanilic acid of 4g/L and a nitrite standard solution of 0.025-1.25 mg/L into a colorimetric tube with a plug of 5mL, shaking up, standing for 5-10 min, diluting with deionized water to 4mL, shaking up, standing for 10-15 min, measuring absorbance A at a wavelength of 530nm, and drawing a standard curve by taking the nitrite concentration as a horizontal coordinate and the A as a vertical coordinate to obtain a regression equation.
(2) Sample processing
(3) And (3) sample determination: adding 400 mu L of sulfanilic acid of 4g/L into a colorimetric tube with a plug of 5mL, adding the sample determination liquid prepared in the step (2), shaking uniformly, standing for 5-10 min, diluting with deionized water to 4mL, shaking uniformly, standing for 10-15 min, determining absorbance A at the wavelength of 530nm, substituting into the regression equation in the step (1), and calculating the nitrite content of the sample.
The preparation method of the copper-iodine doped carbon dots comprises the following steps: weighing 1g of copper acetate, 5-8 g of 3, 5-diiodo-DL-tyrosine and dissolving in 400-600 mL of ultrapure water, uniformly mixing, adding 1-1.5 mL of ethylenediamine, carrying out ultrasonic treatment for 10-20 min, transferring to a polytetrafluoroethylene reaction kettle, heating at 200 ℃ for 10-12 h, naturally cooling, filtering with a filter membrane with the aperture of 0.22 mu m, and carrying out dialysis treatment for 24h with a dialysis bag with the molecular weight cutoff of 3000Da to obtain the water-soluble Cu-I/CDs.
The modified active carbon is as follows: taking activated carbon, adding HCl or HNO of 0.1mol/L3Soaking in the solution for 2h, shaking at room temperature for 12-20 h, filtering, soaking in 0.1mol/L potassium permanganate for 2h, filtering, washing with distilled water until the surface of the activated carbon is neutral, vacuum filtering, and drying at 100 deg.C for 3-6 h to obtain modified activated carbon, wherein the modified activated carbon is obtained from the sample solutionThe weight ratio is 0.5-3: 10.
the invention has the advantages that:
1. the invention utilizes the characteristic of the copper-iodine doped carbon dot to accelerate the diazotization reaction of nitrite and sulfanilic acid and the coupling reaction of the nitrite and the naphthyl ethylenediamine hydrochloride, thereby improving the detection sensitivity by 7 to 8 times.
2. In the detection of sample blood, the interference is greatly reduced and the detection accuracy is improved by two times of treatment through a coprecipitation method, when a food sample is measured, the activated carbon is treated by acid and an oxidant, so that the surface of the activated carbon is activated to form groups, meanwhile, impurities in the activated carbon are cleaned, more pores are exposed, and in the measurement of nitrite of an actual sample, a matrix pigment can be well adsorbed, ascorbic acid in the matrix is effectively removed, and the detection sensitivity and the repeatability of nitrite are effectively improved.
3. The nitrite measuring method established by the invention can meet the requirements of measuring low-content nitrite in blood and measuring nitrite in food, the detection limit of the method can reach 0.01mg/kg, and the RSD is less than 4 percent, and the results show that the technology of the invention is higher than the prior national standard method.
Drawings
FIG. 1 is a standard nitrite absorption spectrum.
FIG. 2 is a standard nitrite linear regression equation.
Detailed Description
The technical solutions of the present invention will be described in further detail with reference to specific examples, but the scope of the present invention is not limited thereto.
Example 1: determination of nitrite in blood samples
1. Preparing copper-iodine doped carbon dots: weighing 1g of copper acetate and 5g of 3, 5-diiodo-DL-tyrosine, dissolving the copper acetate and the 5g of 3, 5-diiodo-DL-tyrosine in 400mL of ultrapure water, uniformly mixing, adding 1mL of ethylenediamine, carrying out ultrasonic treatment for 10min, transferring the mixture into a polytetrafluoroethylene reaction kettle, heating the polytetrafluoroethylene reaction kettle at 200 ℃ for 10h, naturally cooling, filtering the mixture by using a filter membrane with the aperture of 0.22 mu m, and then carrying out dialysis treatment for 24h by using a dialysis bag with the molecular weight cutoff of 3000Da to obtain the water-soluble Cu-I/CDs.
2. Making a nitrite working curve: adding 400 mu L of sulfanilic acid of 4g/L and nitrite standard solution of 0.025-1.25 mg/L into a colorimetric tube with a plug of 5mL, shaking up, standing for 5-10 min, diluting with deionized water to 4mL, shaking up, standing for 10-15 min, measuring absorbance A at the wavelength of 530nm, drawing a standard curve by taking the concentration of the nitrite as a horizontal coordinate and taking A as a vertical coordinate, and obtaining an absorption spectrum shown in a graph 1 to obtain a regression equation, a correlation coefficient, a relative standard deviation, a linear range and the like shown in a graph 1.
TABLE 1 Linear equation, correlation coefficient, relative standard deviation, Linear Range
3. Determination of nitrite in blood samples
(1) Sample treatment: centrifuging 5mL of fresh blood in a centrifuge tube with an anticoagulant at 6000 rpm for 5 min, taking 2mL of supernatant, adding a protein precipitate consisting of 1mL of 0.2 mol/L NaOH and 1mL of 10% zinc sulfate and a blood decolorizing agent, mixing for 30-60 seconds in a vortex manner, centrifuging at 6000 rpm for 5 min, and transferring the supernatant into another centrifuge tube; adding 1mL of deionized water into the precipitate, adding 0.5 mL of 1mol/L HCl, swirling for 30-60 seconds until the precipitate is dissolved, adding 0.5 mL of 0.2 mol/L NaOH and 0.5 mL of 10% zinc sulfate, swirling for 30-60 seconds, centrifuging at 6000 rpm for 5 minutes, taking the supernatant, combining the supernatant with the supernatant obtained by the first treatment, and obtaining a sample to be detected.
(2) Determination of nitrite in sample: adding 400 mu L of sulfanilic acid of 4g/L into a colorimetric tube with a plug of 5mL, adding 2mL of the sample determination solution prepared in the step (1), shaking up, standing for 5-10 min, diluting with deionized water to 4mL, shaking up, standing for 10-15 min, measuring absorbance A at a wavelength of 535 nm, substituting into the regression equation in the step (1), and calculating the nitrite content of the sample to be 0.035 mg/L.
(3) Recovery and precision experiments: respectively adding 3 nitrite standard solutions with different concentrations into serum; each concentration is measured in parallel for 3 times, the standard recovery rate is calculated, and the relative standard deviation RSD is calculated, and the result is shown in a table 2; the measured standard recovery rate of nitrite is 96.5% -98.1%, RSD is 1.21% -1.56%, and the method has good accuracy and precision.
TABLE 2 sample nitrite recovery on standard and RSD (n = 3)
Example 2: determination of nitrite in lettuce sample
1. Preparing copper-iodine doped carbon dots: weighing 1g of copper acetate and 8g of 3, 5-diiodo-DL-tyrosine, dissolving the copper acetate and the 3, 5-diiodo-DL-tyrosine in 600mL of ultrapure water, uniformly mixing, adding 1.5mL of ethylenediamine, carrying out ultrasonic treatment for 20min, transferring the mixture into a polytetrafluoroethylene reaction kettle, heating the mixture at 200 ℃ for 12h, naturally cooling, filtering the mixture by using a filter membrane with the aperture of 0.22 mu m, and then carrying out dialysis treatment for 24h by using a dialysis bag with the molecular weight cutoff of 3000Da to obtain the water-soluble Cu-I/CDs.
2. Preparing modified activated carbon: soaking 100g of activated carbon in 0.1mol/L HCl solution for 2h, shaking at room temperature for 15h, filtering, soaking in 0.1mol/L potassium permanganate solution for 2h, filtering, washing with distilled water until the surface of the activated carbon is neutral, and drying at 100 ℃ for 5h after suction filtration to obtain the modified activated carbon.
3. Making a nitrite working curve: the same as example 1;
4. determination of nitrite content in lettuce sample
(1) Sample treatment: accurately weighing 5g of lettuce samples, placing the lettuce samples in a mortar, fully grinding, transferring residues and juice into a beaker, adding deionized water with the mass 10 times that of the lettuce samples, heating at 70 ℃ for 30min, cooling, filtering, transferring filtrate into a 100mL volumetric flask, and fixing the volume of the deionized water to a scale mark to obtain a nitrite extracting solution;
(2) sample purification treatment: taking 10mL of the nitrite extracting solution prepared in the step (i), adding 1g of modified activated carbon, mixing for 1-2 min in a vortex manner, centrifuging for 10min at 3000 r/min, and taking out supernatant to prepare nitrite sample measuring solution;
(3) determination of nitrite in sample: adding 400 mu L of sulfanilic acid of 4g/L into a colorimetric tube with a plug of 5mL, adding 2mL of the sample determination solution prepared in the step (1), shaking up, standing for 5-10 min, diluting with deionized water to 4mL, shaking up, standing for 10-15 min, measuring absorbance A at a wavelength of 535 nm, substituting into the regression equation in the step (1), and calculating the nitrite content of the sample to be 0.38 mg/kg.
Example 3: determination of nitrite content in orange juice sample
1. Preparing copper-iodine doped carbon dots: the same as example 1;
2. preparing modified activated carbon: the same as example 2;
3. making a nitrite working curve: the same as example 1;
4. determination of nitrite content in orange juice sample
(1) Sample treatment: placing 10.0 mL of orange juice sample into a 100mL beaker, adding 5mL of saturated borax solution, uniformly stirring by using a glass rod, draining into a 500 mL beaker, washing with distilled water for 3-4 times, adding 5mL of 220 g/L zinc acetate solution, stirring for 10min, centrifuging, and filtering the supernatant with a 0.22 mu m filter membrane to obtain a sample solution.
(2) Determination of nitrite in sample: the nitrite content of the sample was calculated to be 0.98 mg/kg as in example 1.
The nitrite determination method established by the invention has the advantages of few processing steps, short time, low processing cost and simple and convenient operation, and has stronger advantages particularly in the detection of NO in low-content serum due to high detection sensitivity.
Claims (3)
1. A method for detecting nitrite by improving Griess reagent comprises the following steps:
(1) making a nitrite working curve: adding 400 mu L of sulfanilic acid of 4g/L and a nitrite standard solution of 0.025-1.25 mg/L into a colorimetric tube with a plug of 5mL, shaking up, standing for 5-10 min, diluting with deionized water to 4mL, shaking up, standing for 10-15 min, measuring absorbance A at a wavelength of 535 nm, and drawing a standard curve by taking the nitrite concentration as a horizontal coordinate and taking A as a vertical coordinate to obtain a regression equation;
(2) sample processing
(3) and (3) sample determination: adding 400 mu L of sulfanilic acid of 4g/L into a colorimetric tube with a plug of 5mL, adding the sample determination liquid prepared in the step (2), shaking up, standing for 5-10 min, diluting with deionized water to 4mL, shaking up, standing for 10-15 min, determining the absorbance A at the wavelength of 535 nm, substituting the absorbance A into the regression equation in the step (1), and calculating the nitrite content of the sample.
2. The method for detecting nitrite using improved Griess reagent as claimed in claim 1, wherein: the preparation method of the copper-iodine doped carbon dots comprises the following steps: weighing 1g of copper acetate and 5-8 g of 3, 5-diiodo-DL-tyrosine, dissolving the copper acetate and the 5-diiodo-DL-tyrosine in 400-600 mL of ultrapure water, uniformly mixing, adding 1-1.5 mL of ethylenediamine, carrying out ultrasonic treatment for 10-20 min, transferring the mixture to a polytetrafluoroethylene reaction kettle, heating the mixture at 200 ℃ for 10-12 h, naturally cooling, filtering the mixture by using a filter membrane with the aperture of 0.22 mu m, and then carrying out dialysis treatment for 24h by using a dialysis bag with the molecular weight cutoff of 3000Da to obtain the water-soluble Cu-I/CDs.
3. The method for rapidly detecting nitrite in food according to claim 1, wherein: the modified active carbon is as follows: taking activated carbon, adding HCl or HNO of 0.1mol/L3After soaking in the solution for 2h, shaking for 12-20 h at room temperature, filtering, soaking in 0.1mol/L potassium permanganate for 2h, filtering, washing with distilled water until the surface of the activated carbon is neutral, performing suction filtration, and drying at 100 ℃ for 3-6 h to obtain the modified activated carbon, wherein the weight ratio of the modified activated carbon to the sample solution is 0.5-3: 10.
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