阅读说明：本技术 一种快速荧光检测铬含量的方法 (Method for rapidly detecting chromium content by fluorescence ) 是由 杨健 黄丽娟 房晟忠 夏万永 李琳 于 2021-08-23 设计创作，主要内容包括：本发明公开了一种快速荧光检测铬含量的方法,该方法是采用组胺、酪胺与荧光胺在pH=8的磷酸缓冲溶液中形成荧光衍生物,该衍生物在390nm激发波长下,在488nm处产生强荧光发射,利用衍生物的氮、羟基对Cr(Ⅵ)具有优良亲和力的特性,发生特异的络合作用,Cr(Ⅵ)诱导荧光信号猝灭,构建了铬的荧光探针,可用于样品中Cr(Ⅵ)的荧光检测；在碱性条件下,双氧水将Cr(Ⅲ)氧化成Cr(Ⅵ),可以对样品中进行总铬的测定,本发明方法可以进行铬的形态分析,方法检出限为0.05mg/kg,方法特异性强,其他金属离子无此现象；将本方法应用于环境食品中铬的检测,结果与相关国家标准测定方法相符；方法具有操作简单、灵敏度高、快速等特点。(The invention discloses a method for rapidly detecting chromium content by fluorescence, which is characterized in that histamine, tyramine and fluorescamine are adopted to form a fluorescence derivative in a phosphate buffer solution with the pH =8, the derivative generates strong fluorescence emission at 488nm under the excitation wavelength of 390nm, the characteristic that nitrogen and hydroxyl of the derivative have excellent affinity to Cr (VI) is utilized to generate specific complexation, and the Cr (VI) induces fluorescence signal quenching, so that a chromium fluorescence probe is constructed, and the method can be used for the fluorescence detection of Cr (VI) in a sample; under the alkaline condition, hydrogen peroxide oxidizes Cr (III) into Cr (VI), and the total chromium in a sample can be measured, the method can be used for carrying out morphological analysis on the chromium, the detection limit of the method is 0.05mg/kg, the method specificity is strong, and other metal ions do not have the phenomenon; the method is applied to the detection of chromium in environmental food, and the result conforms to the related national standard determination method; the method has the characteristics of simple operation, high sensitivity, high speed and the like.)

1. A method for rapidly detecting chromium content by fluorescence is characterized by comprising the following steps:

(1) making Cr (VI) working curve

Adding 50-100 muL of fluorescent probe and Cr (VI) standard solution into a 5mL colorimetric tube with a plug, uniformly mixing and standing for 3-5 min, diluting the mixture to 4mL by using deionized water, uniformly mixing, wherein the concentration range of Cr (VI) in the colorimetric tube with the plug is 0.1-25 mg/kg, measuring the fluorescence intensity at a 488nm wavelength under 390nm excitation wavelength, and drawing a standard curve by taking the concentration of Cr (VI) as a horizontal coordinate and the fluorescence intensity as a vertical coordinate to obtain a regression equation;

(2) sample pretreatment

a. Removing insoluble substances from the water sample by a filter membrane of 0.22 mu m;

b. the method comprises the steps of firstly washing soil twice with acetone, then washing with water, then drying at 30-35 ℃, weighing 1.0g of dry soil, and using 90-110 mL of HNO with mass volume concentration of 63%₃And 40-60 mL of H with the mass volume concentration of 30%₂O₂Dissolving, reacting at 150 +/-5 ℃ for 1.5-2.5 h, then removing acid until the acid is nearly dry, removing insoluble substances by using a 0.22-micron polytetrafluoroethylene filter membrane, taking 5mL of filtrate, and adding water to a constant volume of 50mL to be used as a soil sample;

(3) determination of chromium content

Taking 2mL of the sample to be detected in the step (2), adjusting the pH to be = 10-11 by using 0.1mol/L NaOH, and then adding H with the mass concentration of 25-35% into the sample to be detected₂O₂After reacting for 20-30 min, adding 50-100 muL of fluorescent probe, uniformly mixing and standing for 3-5 min, diluting to 4mL of deionized water, uniformly mixing, measuring fluorescence intensity at a 488nm wavelength under 390nm excitation wavelength, substituting into the regression equation in the step (1), and calculating the total chromium content in the sample;

and (3) adding 50-100 muL of fluorescent probe into 2mL of the sample to be detected in the step (2), uniformly mixing and standing for 3-5 min, diluting the sample to 4mL with deionized water, uniformly mixing, measuring the fluorescence intensity at the position of 488nm wavelength under 390nm excitation wavelength, substituting the fluorescence intensity into the regression equation in the step (1), and calculating to obtain the Cr (VI) content in the sample, wherein the Cr (III) content is the difference value between the total chromium content and the Cr (VI) content.

2. The method for rapid fluorescence detection of chromium content according to claim 1, wherein: the fluorescent probe is prepared by adding 5-7 mL of fluorescamine with the mass volume concentration of 0.02%, 3-5 mL of phosphoric acid buffer solution with the pH =8 and 1-3 mL of deionized water into 3-5 mL of histamine-tyramine mixed solution, carrying out vortex mixing for 1-2 min, and standing for 5-10 min; wherein the mixed solution of histamine and tyramine is prepared by mixing a histamine solution with the concentration of 5mmol/L and a tyramine solution with the concentration of 5mmol/L according to the volume ratio of 2: 1-0.5.

3. The method for rapid fluorescence detection of chromium content according to claim 1, wherein: step (3) H₂O₂The addition amount of (A) is 1-5% of the mass of the sample to be detected.

Technical Field

The invention belongs to the technical field of chemical analysis and detection, and particularly relates to a method for rapidly detecting chromium content by fluorescence.

Background

Chromium is widely present in the atmosphere, water bodies, sediments and organisms, and chromium (Cr) mainly exists in Cr (III) and Cr (VI). The positive significance and the toxicity of the chromium existing in the organism are related to the valence state of the chromium, and the Cr (III) has great effect on maintaining the metabolism of glucose, lipid and protein and is one of trace elements necessary for the organism; cr (VI) is an element having a toxic effect on biological systems, and is 100-fold and 1000-fold more toxic than Cr (III). In recent years, with the rapid development of industry and the increasing of human activities, the chromium pollution in water environment is mostly caused by the random discharge of a large amount of waste water and solid residues and other wastes. However, it should be noted that the pollution caused by chromium is difficult to be reduced or degraded, and generally, the pollution can only be removed or degraded by mutual migration in different areas or different environments, during the migration process, the chromium inevitably undergoes some morphological or valence changes due to changes in environmental conditions, and the chromium with low concentration is gradually enriched into chromium with high concentration, and is absorbed by different levels of organisms in the food chain to cause accumulation of different levels, thereby restricting the development of aquaculture industry and causing unpredictable damage to human health.

At present, the national standard GB/T7467-1987 diphenylcarbodihydrazide spectrophotometry for measuring hexavalent chromium in water only measures hexavalent chromium. The fluorescence analysis method has the characteristics of high sensitivity, rapid determination, visualization and the like, the fluorescence analysis of chromium is reported a lot, and related fluorescence probes mainly comprise fluorescence quantum dots and the like. Fluorescamine itself is not fluorescent with the hydrolysate, but produces similar, strongly fluorescent compounds with primary amino group-containing compounds (including proteins, peptides, amino acids, and various other polypeptides), respectively. The fluorescent derivative mixture formed by fluorescamine, histamine and tyramine is utilized to improve the recognition of Cr (VI), and compared with the derivative formed by fluorescamine and single biogenic amine, the specificity and the stability of the derivative are improved, mainly because of the specificity of the chemical bond binding sites formed by Cr and two fluorescent derivatives, the problem that the interference is the difficult point to be solved by fluorescence analysis is solved.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a method for rapidly detecting the chromium content by fluorescence, which adopts histamine, tyramine and fluorescamine to form a fluorescence derivative in a phosphate buffer solution with the pH =8, the derivative generates strong fluorescence emission at 488nm under the excitation wavelength of 390nm, and utilizes the characteristic that nitrogen and hydroxyl of the derivative have excellent affinity to Cr (VI) to generate specific complexation, and the Cr (VI) induces fluorescence signal quenching to construct a chromium fluorescence probe, so that the method can be used for the fluorescence detection of the Cr (VI) in a sample; under the alkaline condition, hydrogen peroxide oxidizes Cr (III) into Cr (VI) to measure the total chromium content, and the method can also carry out morphological analysis on chromium; the detection limit of the method is 0.05mg/kg, the method has strong specificity, and other metal ions do not have the phenomenon; the method is applied to the detection of chromium in environmental food, and the result conforms to the related national standard determination method; the method has the characteristics of simple operation, high sensitivity, high speed and the like.

The method for rapidly detecting the chromium content by fluorescence is characterized by comprising the following steps of:

(1) making Cr (VI) working curve

Adding 50-100 muL of fluorescent probe and Cr (VI) standard solution into a 5mL colorimetric tube with a plug, uniformly mixing and standing for 3-5 min, diluting the mixture to 4mL by using deionized water, uniformly mixing, wherein the concentration range of Cr (VI) in the colorimetric tube with the plug is 0.1-25 mg/kg, measuring the fluorescence intensity at a 488nm wavelength under 390nm excitation wavelength, and drawing a standard curve by taking the concentration of Cr (VI) as a horizontal coordinate and the fluorescence intensity as a vertical coordinate to obtain a regression equation;

(2) sample pretreatment

a. Removing insoluble substances from the water sample by a filter membrane of 0.22 mu m; the water body samples comprise tap water, lake water, seawater and the like;

b. the method comprises the steps of firstly washing soil twice with acetone, then washing with water, then drying at 30-35 ℃, weighing 1.0g of dry soil, and using 90-110 mL of HNO with mass volume concentration of 63%₃And 40-60 mL of H with the mass volume concentration of 30%₂O₂Dissolving, reacting at 150 +/-5 ℃ for 1.5-2.5 h, then removing acid until the acid is nearly dry, removing insoluble substances by using a 0.22-micron polytetrafluoroethylene filter membrane, taking 5mL of filtrate, and adding water to a constant volume of 50mL to be used as a soil sample;

(3) determination of Total chromium

Taking 2mL of the sample to be detected in the step (2), adjusting the pH to be = 10-11 by using 0.1mol/L NaOH, and then adding H with the mass concentration of 25-35% into the sample to be detected₂O₂After reacting for 20-30 min, adding 50-100 muL of fluorescent probe, uniformly mixing and standing for 3-5 min, diluting to 4mL of deionized water, uniformly mixing, measuring fluorescence intensity at a 488nm wavelength under 390nm excitation wavelength, substituting into the regression equation in the step (1), and calculating the total chromium content in the sample;

said H₂O₂The addition amount of (A) is 1-5% of the mass of a sample to be detected;

and (3) adding 50-100 muL of fluorescent probe into 2mL of the sample to be detected in the step (2), uniformly mixing and standing for 3-5 min, diluting the sample to 4mL with deionized water, uniformly mixing, measuring the fluorescence intensity at the position of 488nm wavelength under 390nm excitation wavelength, substituting the fluorescence intensity into the regression equation in the step (1), and calculating to obtain the Cr (VI) content in the sample, wherein the Cr (III) content is the difference value between the total chromium content and the Cr (VI) content.

The fluorescent probe is prepared by adding 5-7 mL of fluorescamine with the mass volume concentration of 0.02%, 3-5 mL of phosphoric acid buffer solution with the pH =8 and 1-3 mL of deionized water into 3-5 mL of histamine-tyramine mixed solution, carrying out vortex mixing for 1-2 min, and standing for 5-10 min; the mixed solution of histamine and tyramine is prepared by mixing a histamine solution with the concentration of 5mmol/L and a tyramine solution with the concentration of 5mmol/L according to the volume ratio of 2: 1-0.5.

The invention has the advantages that:

1. the invention utilizes a fluorescence derivative mixture formed by fluorescamine, histamine and tyramine to establish a fluorescence probe detection platform, and establishes a new high-sensitivity and strong-selectivity Cr (VI) detection method based on the selective quenching effect of Cr (VI) on the fluorescence probe established by the invention;

2. by introducing two biogenic amines of histamine and tyramine, the formed fluorescent derivative increases the specificity of Cr (VI) combination, and simultaneously, the stability is also improved;

3. the method can be used for carrying out morphological analysis on Cr, has important significance for evaluating the migration characteristic of the chromium, is simple, rapid and sensitive to operate, and is suitable for industrial production and market popularization and application.

Drawings

FIG. 1 is a fluorescence spectrum of quenching of a fluorescent probe by Cr (VI);

FIG. 2 is a linear regression equation of Cr (VI) quenching on fluorescent probes;

FIG. 3 shows the results of the interference experiment, where Blank indicates that no substance was added to the assay system.

Detailed Description

The present invention is further illustrated by the following figures and examples, without limiting the scope of the invention thereto, wherein the process is carried out in a conventional manner unless otherwise specified, and wherein reagents are used, such as reagents used or formulated in a conventional manner, unless otherwise specified.

Example 1: determination of Cr (VI) and Cr (III) in environmental water sample

1. Preparation of fluorescent probe: mixing 2mL of 5mmol/L histamine solution with 1mL of 5mmol/L tyramine solution, adding 5mL of fluorescamine with the mass volume concentration of 0.02%, 3mL of phosphate buffer solution with pH =8 and 1mL of deionized water, mixing for 1min in a vortex manner, and standing for 5min to obtain a fluorescent probe solution;

2. making a Cr (VI) working curve: adding 100 muL of fluorescent probe and Cr (VI) standard solution into a 5mL colorimetric tube with a plug, uniformly mixing and standing for 3min, diluting the mixture to 4mL by using deionized water, uniformly mixing, wherein the concentration range of Cr (VI) in the colorimetric tube with the plug is 0.1-25 mg/kg, measuring the fluorescence intensity at the position of 488nm wavelength under 390nm excitation wavelength, drawing a standard curve by using the Cr (VI) concentration as a horizontal coordinate and the fluorescence intensity as a vertical coordinate to obtain a regression equation, a correlation coefficient, a relative standard deviation, a linear range and the like which are shown in tables 1 and 2;

TABLE 1 Linear equation, correlation coefficient, relative standard deviation, Linear Range

；

3. Determination of Cr (VI) and Cr (III) content in environmental water sample

(1) And (3) determining the content of Cr (VI): placing 2mL of lake water sample treated by a 0.22-micrometer filter membrane into a 5mL colorimetric tube with a plug, adding a 100-mu L fluorescent probe, uniformly mixing and standing for 3min, diluting to 4mL by using deionized water, uniformly mixing, measuring fluorescence intensity at a 488nm wavelength under a 390nm excitation wavelength, substituting into the regression equation in the step 2, and calculating that the Cr (VI) content of the sample is 0.15 mg/kg;

(2) determination of the total chromium content and of Cr (III): taking 2mL of lake water sample treated by a 0.22-micron filter membrane, adjusting the pH value of the lake water sample to 10-11 by using 0.1mol/L NaOH, and then adding H with the mass concentration of 25% into a sample to be detected₂O₂Reacting for 25min by 100 mu L, adding a 100 mu L fluorescent probe, uniformly mixing and standing for 3min, diluting to 4mL by using deionized water, uniformly mixing, measuring fluorescence intensity at a wavelength of 488nm under an excitation wavelength of 390nm, substituting into a regression equation in the step 2, and calculating that the total chromium content of the sample is 0.41mg/kg and the Cr (III) content is 0.26 mg/kg;

(3) recovery and precision experiments: respectively adding 3 Cr (VI) standard solutions with different concentrations into a water sample; 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 Cr (VI) is 98.2-101.6%, RSD is 1.01-1.68%, and the method has good accuracy and precision;

TABLE 2 samples Cr: () Recovery and RSD (n = 3)

；

(4) Method specificity investigation: the potential interfering substance with the concentration of 10mg/kg replaces Cr (VI) with the concentration of 1mg/kg to verify the specificity of the detection system, and FIG. 3 shows that Cr³⁺、Cd²⁺、Co²⁺、Hg²⁺、Cu²⁺、Zn²⁺、Fe²⁺、Fe³⁺、Ca²⁺、Na⁺、K⁺、Mg²⁺、Ni²⁺The results of the influence on the fluorescence signal quenching show that only Cr (VI) induces the fluorescence signal quenching, and the Cr (VI) has better selection specificity on a fluorescence probe system;

(5) the analysis of the certified standard substances, the total chromium and Cr (VI) standard determination purchased from the center of the standard substances is carried out by using the method of the invention, and the result is shown in Table 3;

TABLE 3 analysis results of certified reference substances

。

Example 2: determination of Cr (VI) and Cr (III) in soil

1. Preparation of fluorescent probes

Mixing 3mL of 5mmol/L histamine solution with 1mL of 5mmol/L tyramine solution, then adding 6mL of fluorescamine with the mass volume concentration of 0.02%, 4mL of pH =8 phosphoric acid buffer solution and 2mL of deionized water, mixing for 2min in a vortex mode, and standing for 8min to obtain a fluorescent probe solution;

2. the Cr (VI) working curve was prepared as in example 1;

3. determination of Cr (VI) and Cr (III) content in soil sample

(1) Sample treatment: 5g of soil were weighed, washed twice with 50mL of acetone, then with water and dried at 30 ℃ and 1.0g of dried soil was weighed out and washed with 100mL of HNO₃(63%, m/v) and 50mL H₂O₂(30%, m/v), reacting for 2h at 150 +/-5 ℃, then driving acid to be nearly dry, removing insoluble substances by using a 0.22 mu m polytetrafluoroethylene filter membrane, taking 5mL of filtrate, and fixing the volume to 50mL by using water to be used as a soil sample solution;

(2) and (3) determining the content of Cr (VI): placing 2mL of the sample solution obtained in the step (1) into a 5mL colorimetric tube with a plug, adding 100 muL of a fluorescent probe, uniformly mixing and standing for 4min, diluting to 4mL with deionized water, uniformly mixing, measuring fluorescence intensity at a 488nm wavelength under an excitation wavelength of 390nm, substituting into the regression equation obtained in the step 2, and calculating that the Cr (VI) content of the sample is 111.5 mg/kg;

(3) determination of the total chromium content and Cr (III): taking 2mL of the sample solution in the step (1), adjusting the pH value to 10-11 by using 0.1mol/L NaOH, and adding H with the mass concentration of 30%₂O₂ And reacting for 30min by 100 mu L, adding a 100 mu L fluorescent probe, uniformly mixing and standing for 4min, diluting to 4mL by using deionized water, uniformly mixing, measuring the fluorescence intensity at the wavelength of 488nm under the excitation wavelength of 390nm, substituting into the regression equation in the step 2, and calculating that the total chromium content of the sample is 321.9mg/kg and the Cr (III) content is 210.4 mg/kg.

Example 3: determination of chromium content in tap water samples

1. Preparation of fluorescent probes

Mixing 4mL of 5mmol/L histamine solution with 1mL of 5mmol/L tyramine solution, adding 7mL of fluorescamine with the mass volume concentration of 0.02%, 5mL of phosphoric acid buffer solution with the pH =8 and 1.5mL of deionized water, mixing for 1min in a vortex mode, and standing for 10min to obtain a fluorescent probe solution;

2、Cr（) Manufacturing a working curve: the same as example 1;

3. determination of Cr (VI) and Cr (III) contents in tap water: in the same manner as in example 1, Cr (VI) and Cr (III) were not detected;

cr (b) established by the invention) And Cr (c)) The content determination method has the characteristics of simple operation, high sensitivity, rapidness and the like, does not need large instruments and equipment, and has stronger advantages in actual detection.