A method for detecting cadmium content in rice based on enzyme inhibition and fluorescent inner filter effect

Abstract

The invention discloses a method for detecting cadmium content in rice based on enzyme inhibition and fluorescent inner filter effect, and belongs to the field of rapid detection of heavy metals in food. The method for detecting cadmium content in rice based on enzyme inhibition and fluorescent inner filter effect of the invention comprises the following steps: constructing an up-conversion fluorescent material modified by ethanolamine phosphate, using horseradish peroxidase enzymatic product for fluorescence quenching, according to the inhibition of horseradish peroxidase activity by cadmium element, based on the fluorescent inner filter effect between horseradish peroxidase enzymatic product and up-conversion material, analyzing the quantitative relationship model between fluorescence intensity and cadmium element concentration, and calculating the cadmium content in the test solution according to the model. The method has a stable quantitative detection range and detection limit, the detection range can be maintained at 0.003-6 mg / kg, the detection limit can reach 0.001 mg / kg, the accuracy is high, and the content of cadmium element in rice can be specifically detected.

Description

Technical Field

[0001] The invention relates to a method for detecting cadmium content in rice based on enzyme inhibition and fluorescent inner filtering effect, and belongs to the technical field of rapid detection of heavy metals in food. Background Art

[0002] Long-term consumption of excessive amounts of rice with excessive cadmium can cause irreversible damage to the human body, directly leading to a decline in liver and other body functions. It is reported that when the cadmium content in rice is between 210 and 308 ng / kg, it will become biologically toxic, and when the content reaches 308 ng / kg, the harm to organisms is most significant.

[0003] Common detection methods such as graphite furnace atomic absorption spectroscopy, inductively coupled plasma emission spectroscopy, atomic absorption spectroscopy and other detection technologies have complex sample processing processes, expensive equipment and require professional technicians to operate.

[0004] In order to solve the above-mentioned problem of cadmium detection in rice, the construction of chemical / biosensors for cadmium detection has been increasingly used, mainly including new rapid detection methods such as electrochemical sensors, colorimetric sensors and fluorescence sensors. This type of detection method has the advantages of rapidity and sensitivity, but there are still some problems in the application process, such as photobleaching, background interference elimination, unstable fluorescence signals and low target labeling efficiency. Summary of the invention

[0005] [Technical issues]

[0006] In the existing technology, cadmium element detection has problems such as complex sample processing process, unstable fluorescence signal, photobleaching phenomenon and other factors that lead to reduced detection accuracy.

[0007] [Technical solution]

[0008] In order to solve the above technical problems existing in the prior art, the present invention provides a method for detecting cadmium content in rice based on enzyme inhibition and fluorescent inner filter effect. The method is based on the fluorescent inner filter effect produced by horseradish peroxidase, 3,3',5,5'-tetramethylbenzidine and ethanolamine phosphate modified upconversion nanomaterials to perform specific and rapid detection of cadmium in rice. In order to solve the application problems of the above chemical / biological sensors, an ethanolamine phosphate modified upconversion nanomaterial-horseradish peroxidase-3,3',5,5'-tetramethylbenzidine reaction system is used. Based on the Cd 2+ The purpose of rapid detection of cadmium in rice was achieved by inhibiting the activity of horseradish peroxidase and passivating the fluorescent inner filter effect. This method has a short detection time, strong specificity and high detection sensitivity.

[0009] The object of the present invention is to provide a method for detecting cadmium content in rice based on enzyme inhibition and fluorescent inner filter effect, the method comprising the following steps:

[0010] (1) Pretreatment of samples to be tested

[0011] The crushed rice sample powder is placed in a polytetrafluoroethylene digestion tube, and nitric acid and H2O2 are added and sealed, and then put into microwave digestion. After the digestion is completed, an acid removal instrument is used for acid removal treatment, and then the sample is cooled to room temperature, transferred with ultrapure water, fixed to volume, and filtered through a water membrane to obtain the test solution;

[0012] (2) Ethanolamine phosphate modified upconversion nanomaterials

[0013] The ethanolamine phosphate and the up-conversion material are dissolved in a mixed solution of ethanol, chloroform and water, and the pH value is adjusted to 2.5-3 with hydrochloric acid, and the mixture is stirred for reaction; after the reaction is completed, the reactant is washed with an ethanol aqueous solution and dried to obtain the product;

[0014] (3) Detection of cadmium in the test solution

[0015] A series of rice matrix cadmium standard solutions with different concentrations are prepared, and the rice matrix cadmium standard solutions with different concentrations and the test solution of step (1) are taken respectively, the pH is adjusted, and the ethanolamine phosphate modified up-conversion nanomaterial prepared in step (2) is added, and then horseradish peroxidase is added for reaction, and then 3,3',5,5'-tetramethylbenzidine is added to form a reaction system; after the reaction is completed, the fluorescence signal intensity is measured, and the cadmium element concentration in the rice matrix cadmium standard solutions with different concentrations is used as the horizontal coordinate and the corresponding fluorescence signal intensity is used as the vertical coordinate to construct a quantitative relationship model, and the cadmium element content in the test solution is calculated according to the quantitative relationship model and the fluorescence signal intensity of the test solution.

[0016] In one embodiment, the mass fraction of the nitric acid in step (1) is 65-70%; the mass fraction of H2O2 is 30%; and the volume ratio of nitric acid to H2O2 is 5-8:2, ml:ml.

[0017] In one embodiment, the time of the acid-eliminating treatment in step (1) is 40 to 60 minutes.

[0018] In one embodiment, the mass ratio of the ethanolamine phosphate to the up-conversion material in step (2) is 1:25 to 1:20.

[0019] In one embodiment, the volume ratio of ethanol, chloroform and water in the mixed solution of ethanol, chloroform and water in step (2) is 2:4:3 to 3:4:4.

[0020] In one embodiment, the concentration of the hydrochloric acid in step (2) is 1 mol / L.

[0021] In one embodiment, the ethanol aqueous solution in step (2) is an ethanol aqueous solution with a mass fraction of 40 to 60%.

[0022] In one embodiment, the drying conditions in step (2) are: drying in an oven at 60 to 80° C. for 6 to 8 hours.

[0023] In one embodiment, the upconversion material in step (2) is a lanthanide upconversion nano-solid material.

[0024] In one embodiment, the specific preparation process of the lanthanide upconversion nano solid material is as follows:

[0025] 1) Dissolve ytterbium chloride hexahydrate, yttrium chloride hexahydrate, erbium chloride hexahydrate and gallium chloride hexahydrate in methanol, transfer to a three-necked flask, add oleic acid and 1-octadecene, mix and heat to 150-160° C. for 20-30 minutes, and stir magnetically; then add a methanol solution containing NH4F and NaOH dropwise to the three-necked flask; after the addition is complete, heat in a water bath at 60-70° C. for 40-60 minutes, then quickly heat to 300-350° C., and continue magnetic stirring for 1-2 hours to obtain a product;

[0026] 2) washing the product obtained in step 1) with ethanol aqueous solution, centrifuging, collecting the precipitate, and drying to obtain the product.

[0027] In one embodiment, the pH of step (3) is adjusted by using sodium hydroxide solution to adjust the pH value of the solution to 7.

[0028] In one embodiment, the reaction system of step (3) is to add 400 μL of 0.1 mg / mL ethanolamine phosphate modified upconversion nanomaterial to 1 ml of the test solution, react with 10 μL of horseradish peroxidase for 10 to 15 minutes, and then add 400 μL of 3,3',5,5'-tetramethylbenzidine for 10 to 13 minutes.

[0029] Another object of the present invention is to provide an application of the above-mentioned method in the rapid detection of cadmium content.

[0030] [Beneficial Effects]

[0031] (1) The linear concentration detection range of the rapid detection method for cadmium content in rice established by the present invention is 0.003-6 mg / kg, with a wide linear detection range, and the corresponding detection limit LOD is 0.001 mg / kg. The graphite furnace atomic absorption spectrometry used in the industry standard NY / T 1100-2006 has a minimum detection limit of 0.008 mg / kg and a quantitative limit of 0.1 mg / kg; this method can achieve rapid detection of cadmium content in rice while maintaining the same detection accuracy as the national standard method;

[0032] (2) The specific detection system for cadmium content in rice constructed by the present invention shows a high transferability to the detection of cadmium in rice, can eliminate the interference of other heavy metal elements, eliminate the phenomenon of weak background fluorescence signal, overcome the shortcomings of traditional detection methods, and achieve high specificity and high sensitivity detection of cadmium content in rice. The detection steps are reasonable, the operation is simple, and it has a wide detection linear range and a low detection limit, which makes the method have good practical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a schematic diagram of the principle of rapid detection of cadmium content in rice based on enzyme inhibition and fluorescent inner filter effect of the present invention;

[0034] Figure 2 This is a transmission electron micrograph of the ethanolamine phosphate modified upconversion nanomaterial prepared in Example 1 of the present invention;

[0035] Figure 3 It is a fluorescence signal spectrum diagram of the specific detection system of the cadmium standard solution of different concentrations in Example 1 of the present invention;

[0036] Figure 4 The fluorescence quantitative relationship curve diagram of the cadmium standard solutions of different concentrations in Example 1 of the present invention;

[0037] Figure 5 It is the fluorescence quantitative relationship curve diagram of the cadmium standard solution with different concentrations in Comparative Example 1;

[0038] Figure 6 Graph showing the fluorescence signal intensities corresponding to different metal ions in Example 1 and Comparative Example 2 of the present invention. DETAILED DESCRIPTION

[0039] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly described below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments; based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0040] The specific implementation modes of the present invention will be further described in detail below in conjunction with the accompanying drawings.

[0041] Example 1

[0042] A method for detecting cadmium content in rice based on enzyme inhibition and fluorescent inner filter effect, the specific steps are:

[0043] S1. Rice digestion treatment:

[0044] (1) Weigh 5 g of the rice to be tested and blank rice and place them in a high-speed pulverizer, grind them at 6000 rpm for 1 minute, and pass them through a 40-mesh sieve; then accurately weigh 0.2000 g of sample powder in a polytetrafluoroethylene digestion tube, slowly add 5.0 mL of 65% nitric acid and 2.0 mL of 30% H2O2, tighten the seal, and put it in a microwave digestion tube; the temperature is raised from 80°C to 200°C and maintained for 5 minutes. After the digestion is completed, use an acid removal instrument to remove the acid at 160°C for 45 minutes, then take it out and cool it to room temperature;

[0045] (2) The cooled digestion solution was transferred several times with ultrapure water and fixed to 10 mL. It was then filtered through a 0.22 μm filter membrane and the filtrate was collected as the test solution and rice matrix solution.

[0046] S2. Preparation of upconversion nano-solid materials modified with ethanolamine phosphate

[0047] (1) Weigh 0.065 g of ytterbium chloride hexahydrate, 0.117 g of yttrium chloride hexahydrate, 0.006 g of erbium chloride hexahydrate and 0.091 g of gallium chloride hexahydrate respectively, dissolve them in 4 mL of methanol (analytical grade), transfer them to a three-necked flask, add oleic acid and 1-octadecene, mix and heat to 160° C. for 30 minutes, and stir magnetically; then add 20 mL of methanol solution containing 0.4446 g of NH4F and 0.3 g of NaOH dropwise to the three-necked flask; after the addition is complete, heat in a water bath at 70° C. for 40 minutes, then quickly heat to 300° C., and continue to stir magnetically for 1 hour; obtain a reaction mixture;

[0048] (2) washing the reaction mixture obtained in step (1) three times with 75 mL of 80% ethanol aqueous solution, then centrifuging at 6000 rpm for 10 minutes to remove unreacted precursor, and drying in a vacuum drying oven at 60° C. for 24 hours to collect upconversion nanoparticle solid powder;

[0049] (3) Weighing 20.8 mg of ethanolamine phosphate and 200 mg of the upconversion nano solid powder prepared in step (2) respectively, dissolving them in 18 mL of a mixed solution of ethanol: chloroform: water, wherein the volume ratio of ethanol, chloroform and water is 2:4:3, and dissolving them by ultrasonic acceleration for 30 minutes; then adding 1 mol / L hydrochloric acid to adjust the pH to 2.5, and stirring vigorously for 1 hour; finally, washing three times with 50% ethanol aqueous solution, and drying in a vacuum drying oven at 60° C. for 6 hours, to obtain an ethanolamine phosphate modified upconversion nano material;

[0050] S3. Construction of standard curves at different cadmium concentrations

[0051] Standard solutions of cadmium concentrations (0, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0 and 6.0 μmol / L) with different rice matrix configurations were prepared, the pH was adjusted to 7 with sodium hydroxide solution, and 400 μL of 0.1 mg / mL ethanolamine phosphate-modified upconversion nanomaterials were added, followed by the addition of 10 μL of horseradish peroxidase for 15 minutes; 400 μL of 3,3',5,5'-tetramethylbenzidine was added and mixed for 13 minutes, and the fluorescence signal intensity was measured using a fluorescence signal meter. A quantitative relationship model was constructed with cadmium element concentration as the horizontal axis and fluorescence signal intensity as the vertical axis.

[0052] S4. Detection of cadmium concentration in rice

[0053] 1 mL of the rice digestion solution prepared above was measured, the pH was adjusted to 7 with sodium hydroxide, and 400 μL of 0.1 mg / mL ethanolamine phosphate-modified upconversion nanomaterials were added, followed by adding 10 μL of horseradish peroxidase for 15 minutes; 400 μL of 3,3',5,5'-tetramethylbenzidine was added and mixed for 13 minutes, and the fluorescence signal intensity was measured; and the cadmium concentration in the rice digestion solution was calculated according to the quantitative relationship model constructed above.

[0054] Result determination:

[0055] Figure 2 This is a transmission electron microscopy image of the upconversion nanomaterial modified with ethanolamine phosphate; Figure 3 is the fluorescence intensity of the standard solution with different cadmium concentrations measured in this example, Figure 4 The quantitative relationship model of the standard curve constructed for the fluorescence intensity and cadmium concentration of different cadmium concentrations, the standard curve equation is: Y = 173.95X-85.254; R 2 The linear concentration detection range is 0.003-6 mg / kg, with a wide linear detection range, and the corresponding detection limit LOD is 0.001 mg / kg.

[0056] Example 2 Addition recovery test

[0057] A series of different concentrations of Cd 2+ The solution was added to a digestion tank containing 0.4 g of rice sample, 2 mL of deionized water and 10 mL of concentrated nitric acid, and digestion treatment and determination were performed using the treatment method of Example 1. The results are shown in Table 1.

[0058] It can be found from Table 1 that the recovery rate of the spiked rice can reach 97.3-108.2%. Compared with the ICP-MS detection method, it can be found that the two detection results are similar, indicating that the detection method of the present invention has good accuracy.

[0059] Table 1. Recovery of cadmium ions spiked in rice

[0060]

[0061] Comparative Example 1

[0062] The only difference from Example 1 is that (3) in S2 is replaced by: weigh 10 mg of upconversion particles and add them into a 100 mL conical flask containing 6 mL of ethanol solution, ultrasonicate for 20 min, then transfer to a 35°C water bath, add 2 mL of deionized water and 0.25 mL of 25% ammonia solution, and react at 5-10°C under magnetic stirring; then, add 0.006 mL of tetraethyl orthosilicate dropwise at 65°C, react for 8 hours, and then add 0.01 mL of 3-aminopropyltriethoxysilane and react for 3 hours; after completion, wash the product with 50% ethanol solution and dry it in a vacuum oven to obtain amino-modified upconversion particles.

[0063] S3. Construction of standard curves for different cadmium concentrations

[0064] Standard solutions with different cadmium concentrations (0, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0 and 6.0 μmol / L) were prepared, the pH was adjusted to 7 with sodium hydroxide solution, 400 μL of 0.1 mg / mL amino-modified upconversion nanomaterials and unmodified upconversion nanomaterials were added respectively, and then 10 μL of horseradish peroxidase was added to react for 15 minutes; then 400 μL of 3,3',5,5'-tetramethylbenzidine was added and mixed for 13 minutes, and the fluorescence signal intensity was measured.

[0065] The results showed that the fluorescence intensity changes of different concentrations of cadmium were not linear. Figure 5 .

[0066] Comparative Example 2

[0067] 1 mL of 5 μmol / L sodium, magnesium, lead, mercury, palladium, iron, calcium and zinc solutions and blank aqueous solution were measured respectively, the pH was adjusted to 7 with sodium hydroxide, and 400 μL of 0.1 mg / mL ethanolamine phosphate modified upconversion nanomaterials were added, followed by 10 μL of horseradish peroxidase for 15 minutes; then 400 μL of 3,3',5,5'-tetramethylbenzidine was added and mixed for 13 minutes to measure the fluorescence signal intensity.

[0068] The results are as follows Figure 6 As shown in the figure, it can be seen from the results that the fluorescence signal intensity of each detection system is equivalent to the blank, and there is no strong fluorescence response value; this shows that the system has no response to sodium, magnesium, lead, mercury, palladium, iron, calcium and zinc metal ions, indicating that the detection method of the present invention has a specific response to cadmium.

Claims

1. A method for detecting cadmium content in rice based on enzyme inhibition and fluorescent inner filter effect, characterized in that: The method comprises the following steps: (1) Pretreatment of samples to be tested The crushed rice sample powder is placed in a polytetrafluoroethylene digestion tube, and nitric acid and H2O2 are added and sealed, and digested. After the digestion is completed, an acid removal instrument is used to remove the acid, and the sample is cooled to room temperature, transferred, fixed to volume, and filtered to obtain the test solution. (2) Ethanolamine phosphate modified upconversion nanomaterials The ethanolamine phosphate and the up-conversion material are dissolved in a mixed solution of ethanol, chloroform and water, and the pH value is adjusted to 2.5-3 with hydrochloric acid, and the reaction is stirred; then the reactant is washed with an ethanol aqueous solution and dried to obtain the up-conversion material; the up-conversion material is a lanthanide up-conversion nano solid material; (3) Detection of cadmium in the test solution A series of rice matrix cadmium standard solutions of different concentrations are prepared, and the rice matrix cadmium standard solutions of different concentrations and the test solution of step (1) are taken respectively, the pH is adjusted to neutral, and the ethanolamine phosphate modified up-conversion nanomaterial prepared in step (2) is added, and then horseradish peroxidase is added for reaction, and then 3,3',5,5'-tetramethylbenzidine is added to form a reaction system; after the reaction is completed, the fluorescence signal intensity is measured, and the cadmium element concentration in the rice matrix cadmium standard solutions of different concentrations is used as the horizontal axis and the corresponding fluorescence signal intensity is used as the vertical axis to construct a quantitative relationship model, and the cadmium element content in the test solution is calculated according to the quantitative relationship model and the fluorescence signal intensity of the test solution.

2. The method according to claim 1, characterized in that In step (2), the mass ratio of the ethanolamine phosphate to the upconversion material is 1:25 to 1:

20.

3. The method according to claim 1, characterized in that The volume ratio of ethanol, chloroform and water in the mixed solution of ethanol, chloroform and water in step (2) is 2:4:3 to 3:4:

4.

4. The method according to claim 1, characterized in that: The ethanol aqueous solution in step (2) is an ethanol aqueous solution with a mass fraction of 40-60%.

5. The method according to claim 1, characterized in that The drying conditions in step (2) are: drying in an oven at 60-80°C for 6-8 hours.

6. The method according to claim 1, characterized in that The specific preparation process of the lanthanide upconversion nano solid material is as follows: 1) Dissolve ytterbium chloride hexahydrate, yttrium chloride hexahydrate, erbium chloride hexahydrate and gallium chloride hexahydrate in methanol, transfer to a three-necked flask, add oleic acid and 1-octadecene, mix and heat to 150-160°C for 20-30 minutes, and stir magnetically; then add methanol solution containing NH4F and NaOH dropwise into the three-necked flask; After the addition is completed, heat in a water bath at 60-70° C. for 40-60 minutes, then quickly heat to 300-350° C., and continue magnetic stirring for 1-2 hours to obtain the product; 2) Wash the product obtained in step 1) with ethanol aqueous solution, centrifuge, take the precipitate, and dry to obtain the product.

7. The method according to claim 1, characterized in that The mass fraction of the nitric acid in step (1) is 65-70%; the mass fraction of H2O2 is 30%; and the volume ratio of nitric acid to H2O2 is 5-8:2, ml:ml.

8. The method according to claim 1, characterized in that The reaction system of step (3) is as follows: 400 μL of 0.1 mg / mL ethanolamine phosphate modified upconversion nanomaterials are added to 1 ml of the test solution, 10 μL of horseradish peroxidase is reacted for 10 to 15 minutes, and then 400 μL of 3,3',5,5'-tetramethylbenzidine is added to react for 10 to 13 minutes.

9. Use of the method according to any one of claims 1 to 8 in rapid detection of cadmium content.

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