Method for detecting ammonium salt by using rare earth modified nanocopper

Abstract

A method for detecting ammonium salts using rare earth-modified nano copper, which relates to the technical field of the application of nanomaterials. Take CuGdOx and ammonium chloride with gradient concentrations to obtain 3 groups of parallel samples, a total of 30 groups, and add them to a 96-well enzyme-linked immunosorbent assay (ELISA) plate in sequence; take CuGdOx and the sample to be tested and add them to the blank wells of the 96-well ELISA plate; after standing and mixing for 30 minutes, place it in an ELISA detector to measure the linear relationship between absorbance and concentration, and calculate the ammonium salt concentration of the sample based on this linear relationship. Among them, the drawn linear relationship (standard curve) can be reused for more than one month without retesting, and the error does not exceed 5%. While being sensitive in detection, it is easy to promote, has low cost, and can be detected in batches.

Description

Technical Field

[0001] The present invention relates to the technical field of the application of nanomaterials, and particularly to a method for detecting ammonium salts by using rare earth modified nanocopper. Background Art

[0002] With the development of science and technology, the relatively backward ammonia nitrogen monitoring technology has brought a series of ecological safety and food safety problems. For example, the random discharge of ammonia nitrogen wastewater and waste liquid has led to an increase in nitrite in water bodies, and the addition of melamine to infant milk powder has led to malformations in children's development.

[0003] For the detection of ammonia nitrogen, the earliest method used was the Kjeldahl method that emerged in 1883. To date, after the improvement of scholars in successive generations, a variety of detection methods have been developed, such as the Nessler's reagent method, the distillation neutralization titration method, the salicylic acid spectrophotometry method, etc. However, they all have various deficiencies. For example: 1. The Kjeldahl method has bulky instruments and cannot distinguish ammonia, ammonium salts, and nitrogen in proteins. For the interference of various proteins, the accurate concentration of ammonium salts cannot be measured. 2. The Nessler's reagent method produces a large amount of waste liquid, and heavy metal mercury is used in the method, which is prone to generate new pollution. 3. The distillation neutralization titration method produces a large amount of waste liquid and the detection procedure is complex. 4. The salicylic acid spectrophotometry method has a long display time (1 h), and the experimental conditions are relatively harsh. It is not easy to grasp the accuracy. Any deviation in any detail will affect the measurement results.

[0004] The existing methods are not sufficient to meet the complex detection needs. Therefore, there is an urgent need for technological innovation in the detection of ammonia nitrogen such as ammonium salts. Our experimental research found that nanocopper can combine with ammonium salts to form a copper ammonia complex. Due to insufficient coordination, the copper ammonia complex does not show color, while nanocopper itself has a strong absorption peak at 607 nm due to the surface plasmon resonance effect. With the reaction with ammonium salts, the absorption peak weakens. However, conventional nanocopper is not stable in aqueous solution and requires a better method to stabilize nanocopper to facilitate the gentle reaction of ammonium salts with nanocopper, so that the ammonium salt concentration and absorbance show a good linear relationship. The previous solutions such as coating a layer of silica on the surface of nanocopper, and then through literature searches in CNKI and Web of Science and other databases, no successful coating articles have been found. This may be because the conventional tetraethyl orthosilicate hydrolysis method requires a strong alkaline environment, and nanocopper is not stable in aqueous solution, making it impossible to achieve the coating structure. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for detecting ammonium salts by using rare earth modified nanocopper, which is sensitive in detection, easy to promote, low in cost, and can be batch-detected.

[0006] A method for detecting ammonium salts by using rare earth modified nanocopper includes the following steps:

[0007] Prepare Group A samples: Take 100 μL of CuGdOx and 100 μL of ammonium chloride with gradient concentrations to obtain 3 sets of parallel samples, a total of 30 groups, and add them to a 96-well microplate in sequence;

[0008] Prepare Group B samples: Take 100 μL of CuGdOx + 100 μL of the sample to be tested and add it to the blank wells of a 96-well microplate;

[0009] After standing and mixing for 30 minutes, place it in an enzyme-linked immunosorbent assay (ELISA) detector to measure the linear relationship between the absorbance and concentration of Group A samples. Based on this linear relationship, calculate the ammonium salt concentration of the samples.

[0010] Preferably, the content of ammonium chloride with gradient concentrations in the present invention is 0.1 - 1.0 mg / mL.

[0011] Preferably, the process for preparing CuGdOx in the present invention is as follows: Take 760 mg of Gd(acac)3 and 525 mg of Cu(acac)2, ultrasonically disperse them in 50 mL of DEG, then add 1.5 g of PVP, stir magnetically at high speed and heat to 130 °C, and maintain for 20 minutes to form a uniform dark blue transparent solution; Place the solution in a 100 mL reaction kettle, transfer it to a vacuum drying oven at 230 °C, and react for 24 hours; After natural cooling, centrifuge at 13000 rpm to collect, wash three times with water and ethanol, obtain CuGdOx, and after freeze-drying, collect and reserve for use.

[0012] Preferably, in the present invention, 10 mg of CuGdOx is aspirated into a 10 mL centrifuge tube, and 10 mL of water is added to prepare a 100 μg / mL aqueous solution of CuGdOx.

[0013] Preferably, in the present invention, 10 mg of the stock solution of CuGdOx is aspirated into a 10 mL centrifuge tube, 7 mL of water is added, after balancing, it is placed in a centrifuge to centrifuge, the supernatant is removed, and 10 mL of water is added to prepare a 100 μg / mL aqueous solution of CuGdOx.

[0014] Adopting the technical solution of the present invention has the following advantages compared with the prior art:

[0015] 1. Sensitive detection: The ammonium chloride with gradient concentrations in the present invention is selected at 0.1 - 1.0 mg / mL, having excellent linearity. That is, at 20 min, the linearity is 0.98, and with the extension of time, the linearity is better; Moreover, the standard curve can be used for several weeks, and still has 95% accuracy.

[0016] 2. The synthesis of CuGdOx in the present invention is simple, easy to promote, low in cost, requires less sample for detection, only 0.1 mL, the detection is simple and fast, and can be detected batchwise and in large quantities.

[0017] 3. In the detection method of the present invention, the gradient concentration ammonium chloride and CuGdOx used reduce the interference of nitrogen-containing compounds such as urea, and can detect ammonia and ammonium salts more sensitively.

[0018] 4. In the detection method of the present invention, the gradient concentration ammonium chloride and CuGdOx used produce less waste liquid after detection and are easy to recycle. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is a process schematic diagram of the detection method of the present invention.

[0020] Figure 2 is a reaction mechanism diagram of the gradual complexation of copper nuclei in nano-CuGdOx and ammonium salts.

[0021] Figure 3 is a transmission electron microscope image of CuGdOx.

[0022] Figure 4 is a schematic diagram of the linear relationship between the absorbance and concentration of the solution in Group A.

[0023] Figure 5 is a precision broken line graph obtained in Example 3. DETAILED DESCRIPTION OF THE INVENTION

[0024] As Figure 1 shown, a method for detecting ammonium salts using rare earth-modified nano-copper includes the following steps:

[0025] Prepare the solution in Group A: Take 0.1 mL of 100 μL CuGdOx and 0.1 mL of 100 μL of 0.1 - 1.0 mg / mL gradient concentration ammonium chloride to obtain 3 parallel samples, a total of 30 groups, and add them to a 96-well microplate in sequence;

[0026] Prepare the solution in Group B: Take 100 μL of CuGdOx + 100 μL of the sample to be tested and add it to the blank wells of a 96-well microplate;

[0027] After standing and mixing for 30 minutes, place it in an enzyme-labeled detection instrument, measure the linear relationship between the absorbance and concentration of the solution in Group A, and calculate the ammonium salt concentration of the sample based on this linear relationship.

[0028] The process for preparing CuGdOx in the present invention is as follows: Take 760 mg of Gd(acac)3 and 525 mg of Cu(acac)2, ultrasonically disperse them in 50 mL of DEG, then add 1.5 g of PVP, and magnetically heat and stir at high speed to 130 °C for 20 minutes to form a uniform dark blue transparent solution; place the solution in a 100 mL reaction kettle, transfer it to a vacuum drying oven at 230 °C, and react for 24 hours; after natural cooling, centrifuge and collect at 13000 rpm, wash three times with water and ethanol to obtain CuGdOx. After freeze-drying, collect and reserve for use.

[0029] Absorb 10 mg of CuGdOx into a 10 mL centrifuge tube, add 10 mL of water, and prepare a 100 μg / mL aqueous solution of CuGdOx.

[0030] Absorb 10 mg of CuGdOx into a 10 mL centrifuge tube, add 7 mL of water, balance it, place it in a centrifuge to centrifuge, remove the supernatant, add 10 mL of water, and prepare a 100 μg / mL aqueous solution of CuGdOx.

[0031] The detection method of the present invention requires the following substances for the reaction: (1) 0.1 mL of ammonium chloride with gradient concentrations (0.1 - 1.0 mg / mL); (2) 0.1 mg / mL of CuGdOx; (3) the sample to be tested: 0.1 mL; (4) one microplate (96-well plate); (5) one microplate reader.

[0032] The process conditions of the detection method of the present invention include: (1) Microplate reader: supporting 96-well plates; (2) Temperature: the detection temperature is room temperature (10 - 30 °C); (3) Pressure: standard atmospheric pressure; (4) Time: the mixing time is 30 minutes.

[0033] As Figure 2 Shown in the reaction mechanism diagram, nano-CuGdOx has strong stability. The gadolinium oxide shell with a loose structure slows down the reaction between nano-copper and ammonium salt well, showing excellent linearity. After the copper core in nano-CuGdOx gradually complexes with ammonium salt, the overall surface plasmon resonance of nano-copper weakens, the absorption peak at 600 nm decreases, and no new peak is generated. Therefore, the change in ammonium salt concentration and absorbance can be measured by a microplate reader, and the ammonium concentration of the unknown sample can be calculated.

[0034] Example 1:

[0035] 1. Synthesis and storage of CuGdOx: Weigh 760 mg of Gd(acac)3 and 525 mg of Cu(acac)2, ultrasonically disperse them in 50 mL of DEG, then add 1.5 g of PVP, and heat and stir magnetically at high speed to 130 °C for 20 minutes to form a uniform dark blue transparent solution. Place the solution in a 100 mL reaction kettle, transfer it to a vacuum drying oven at 230 °C, and react for 24 hours. After natural cooling, centrifuge at 13000 rpm to collect, wash three times with water and ethanol, and obtain CuGdOx. After freeze-drying, collect and reserve.

[0036] The prepared CuGdOx can be verified by transmission electron microscopy whether it has obtained a coated structure. The transmission electron microscopy images of CuGdOx are as Figure 3 shown. Among them, A is the electron microscopy image of the prepared CuGdOx at a scale of 500 nm, B is the electron microscopy image of the prepared CuGdOx at a scale of 200 nm, C is the electron microscopy image of the prepared CuGdOx at a scale of 100 nm, D is the electron microscopy image of CuGdOx at a scale of 500 nm after sufficient reaction with a sufficient amount of ammonium chloride, E is the electron microscopy image of CuGdOx at a scale of 200 nm after sufficient reaction with a sufficient amount of ammonium chloride, and F is the electron microscopy image of CuGdOx at a scale of 50 nm after sufficient reaction with a sufficient amount of ammonium chloride.

[0037] 2. Preparation of 100 ug / mL CuGdOx solution:

[0038] Weigh 10 mg of CuGdOx into a 10 mL centrifuge tube, add 10 mL of water to prepare a 100 ug / mL aqueous solution of CuGdOx.

[0039] 3. Preparation of the standard curve: Group A: 100 uL of CuGdOx (100 ug / mL aqueous solution) + 100 uL of ammonium chloride with gradient concentrations (0.1 - 1.0 mg / mL). Take 3 parallel samples, a total of 30 groups, and add them to a 96-well plate in sequence;

[0040] 4. Detection of samples: After adding to the wells of the enzyme-linked immunosorbent assay (ELISA) plate in Group A, immediately add in Group B: 100 uL of CuGdOx (100 ug / mL aqueous solution) + 100 uL of the sample to be tested into the blank wells of the 96-well plate. After the two groups are allowed to stand and mix for 30 minutes, place them in an enzyme-labeled detection instrument to measure the linear relationship between the absorbance and concentration of Group A, and calculate the ammonium salt concentration of the sample based on this linear relationship. For example: Through linear fitting, the linear relationship of Group A is obtained as y = ax + b, where x is the ammonium salt concentration and y is the absorbance. Measure the absorbance y1 of the ammonium salt of the unknown sample, and the ammonium salt concentration x1 of the sample can be calculated (calculation method: x1 = (y - b) / a).

[0041] Example 2:

[0042] 1. Synthesis and storage of CuGdOx: Take 760 mg of Gd(acac)3 and 525 mg of Cu(acac)2, ultrasonically disperse them in 50 mL of DEG, then add 1.5 g of PVP, and magnetically stir and heat at high speed to 130 °C for 20 minutes to form a uniform dark blue transparent solution. Place the solution in a 100 mL reaction kettle, transfer it to a vacuum drying oven at 230 °C, and react for 24 hours. After natural cooling, centrifuge at 13000 rpm to collect, wash three times with water and ethanol, obtain CuGdOx, and collect it in about 30 mL of ethanol solution. Ultrasonically disperse the ethanol solution of CuGdOx into a weighed 2 mL centrifuge tube. After centrifugation, suck out the supernatant, dry and quantify it to calculate the concentration of the CuGdOx ethanol solution (mother liquor).

[0043] 2. Preparation of 100 ug / mL CuGdOx solution:

[0044] Absorb the mother liquor containing 10 mg of CuGdOx into a 10 mL centrifuge tube, add 7 mL of water, balance it, place it in a centrifuge to centrifuge, remove the supernatant, add 10 mL of water, and prepare a 100 ug / mL aqueous solution of CuGdOx.

[0045] 3. Preparation of standard curve: Group A: 100 uL of CuGdOx (100 ug / mL aqueous solution) + 100 uL of ammonium chloride with gradient concentrations (0.1 - 1.0 mg / mL). Take 3 parallel samples, a total of 30 groups, and add them to a 96-well plate in sequence;

[0046] 4. Detection of samples: After adding to the wells of the enzyme-labeled plate in Group A, immediately in Group B: 100 uL of CuGdOx (100 ug / mL aqueous solution) + 100 uL of the sample to be tested, add it to the blank wells of a 96-well plate. After the two groups are left standing and mixed for 30 minutes, place them in an enzyme-labeled detection instrument to measure the linear relationship between the absorbance and concentration of the samples in Group A, and calculate the ammonium salt concentration of the sample to be tested based on this linear relationship. The linear relationship between the absorbance and concentration of Group A is as Figure 4 shown, where A is the standard curve of ammonium chloride concentration and absorbance, and B is the R calculated after linear fitting at the corresponding time points in A 2 .

[0047] Example 3:

[0048] 1. Precision verification: Absorb the mother liquor containing 10 mg of CuGdOx into a 10 mL centrifuge tube, add 7 mL of water, balance it, place it in a centrifuge to centrifuge, remove the supernatant, add 10 mL of water, and prepare a 100 ug / mL aqueous solution of CuGdOx.

[0049] 2. Preparation of standard curve: Group C: 100 uL of CuGdOx (aqueous solution at 100 ug / mL) + 100 uL of ammonium chloride with gradient concentrations (0.15, 0.25, -0.95, 1.0 mg / mL). Take 3 sets of parallel samples, a total of 30 sets, and add them to a 96-well plate in sequence. After standing and mixing for 30 minutes, place them in an enzyme-labeled detector to measure the absorbance value;

[0050] Divide the absorbance measured for Group C by the values calculated from the standard curve (0.15, 0.25, -0.95, 1.0 mg / mL), and plot a line graph for the obtained data, as Figure 5 . Figure 5 It shows the precision calculation method adopted in this embodiment and the precision line graph obtained by using the precision calculation method.

[0051]

[0052] As shown in the above table, compared with the Nash reagent spectrophotometry, the CuGdOx enzyme-labeled method of the present invention has very little waste liquid generated and does not generate heavy metals such as mercury under the condition of good precision, and is easy to recycle.

Claims

1. A method for detecting ammonium salts using rare earth-modified nano copper, characterized in that it includes the following steps: Preparing group A samples: Take 100 μL of 0.1 mg / mL CuGdOx and 100 μL of ammonium chloride with gradient concentrations to obtain 3 parallel samples, a total of 30 groups, and add them to a 96-well enzyme-linked immunosorbent assay (ELISA) plate in sequence; the content of ammonium chloride with gradient concentrations is 0.1 - 1.0 mg / mL; Preparing group B samples: Take 100 μL of CuGdOx + 100 μL of the sample to be tested and add it to the blank wells of a 96-well ELISA plate; after standing and mixing for 30 minutes, place it in an enzyme-linked immunosorbent assay detector to measure the linear relationship between the absorbance and concentration of group A samples, and calculate the ammonium salt concentration of the sample based on this linear relationship; Among them, the process for preparing CuGdOx is as follows: Take 760 mg of Gd(acac)3 and 525 mg of Cu(acac)2, ultrasonically disperse them in 50 mL of DEG, then add 1.5 g of PVP, stir magnetically and heat at high speed to 130 °C, and keep for 20 minutes to form a uniform dark blue transparent solution; place the solution in a 100 mL reaction kettle, transfer it to a 230 °C vacuum drying oven, and react for 24 hours; after natural cooling, centrifuge and collect at 13000 rpm, wash three times with water and ethanol to obtain CuGdOx. After freeze-drying, collect and reserve.

2. The method for detecting ammonium salts using rare earth-modified nano copper according to claim 1, characterized in that Absorb 10 mg of CuGdOx into a 10 mL centrifuge tube, add 10 mL of water, and prepare a 100 μg / mL aqueous solution of CuGdOx.

3. The method for detecting ammonium salts using rare earth-modified nano copper according to claim 1, characterized in that Absorb 10 mg of the stock solution of CuGdOx into a 10 mL centrifuge tube, add 7 mL of water, balance it, place it in a centrifuge to centrifuge, remove the supernatant, add 10 mL of water, and prepare a 100 μg / mL aqueous solution of CuGdOx.

Images