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Melamine colorimetric detection method based on enzymatic amino hydrolysis reaction

A technology of melamine and ammonia water, applied in the field of detection, can solve the problems of cumbersome processing and analysis of samples, high-throughput detection of difficult samples, and lack of high-throughput melamine.

Inactive Publication Date: 2013-03-06
SHANGHAI INST OF ORGANIC CHEM CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the methods introduced by these national standards are suitable for the detection of different objects and sample concentrations, expensive laboratory equipment and professional technicians are required to perform tedious processing and analysis of samples, and the above methods can only be used once for each instrument. Measure one sample, it is difficult to perform high-throughput detection of a large number of samples
Due to price and throughput issues, the above methods can only perform relatively accurate measurements on limited samples
[0006] In addition, there is still a lack of high-throughput, low-concentration or very low-concentration melamine detection methods that are easy to operate in the art

Method used

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  • Melamine colorimetric detection method based on enzymatic amino hydrolysis reaction
  • Melamine colorimetric detection method based on enzymatic amino hydrolysis reaction
  • Melamine colorimetric detection method based on enzymatic amino hydrolysis reaction

Examples

Experimental program
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Effect test

Embodiment 1

[0094] Example 1: Genetic modification, expression and purification of aminohydrolase

[0095] Although the natural coding sequence of the aminohydrolase can also be used to achieve recombinant expression, the expression level is low, so in this example, the codon-optimized gene was used for recombinant expression.

[0096] Codon optimization (the optimized nucleotide sequence is shown in SEQ ID NO.: 1 or see Figure 8 , its ORF is located at position 7-1428, and its encoded amino acid sequence is shown in SEQ ID NO.: 2 or see Figure 9). The optimized sequence was prepared by artificial total synthesis, and then connected to the commercially available vector pET21a through BamHI and XhoI sites.

[0097] Using conventional commercially available Escherichia coli BL21 (DE3) as the expression strain, at 16°C, under the induction of 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG), the nitrogen-terminal bands were produced. Recombinant Tri A with 6 histidine (His) tags (see ...

Embodiment 2

[0101] Embodiment 2: sample purification

[0102] (1) Pipette 10mL samples at room temperature (samples include: milk with 5ppm, 10ppm, 15ppm, 50ppm melamine added respectively) to a 50mL centrifuge tube, add 75 μL of anhydrous acetic acid, vortex for 30 seconds, and incubate at room temperature for 5 minutes , add 15 mL of acetonitrile and vortex for 30 seconds. The resulting mixture was centrifuged at 5000 rpm for 10 minutes, and all the supernatant was transferred to another test tube as the sample to be tested.

[0103] (2) After adding 2 mL of methanol to activate the Strata XC SPE column, add 2 mL of distilled aqueous solution containing 60% acetonitrile and 0.5% acetic acid.

[0104] (3) Add the sample to be tested obtained in step (1) into the activated Strata XC SPE column, add 2mL of 0.5% acetic acid aqueous solution, 2mL of 0.5% acetic acid in methanol and 2mL of 100% methanol to wash the column, and finally add 1mL ammonia water methanol solution (the volume rati...

Embodiment 3

[0106] Embodiment 3: Enzymolysis reaction

[0107] (1) Add 100 μ L of the sample to be tested obtained in Example 2 to a 96-well plate;

[0108] (2) Add 5 μ L of the aminohydrolase mixture obtained in Example 1 to each well, and tap the microwell plate to mix evenly;

[0109] (3) Incubate at room temperature for 20 minutes.

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Abstract

The invention relates to a melamine colorimetric detection method based on enzymatic amino hydrolysis reaction, and in particular provides a method for quantitatively detecting melamine in a sample. The method comprises the steps of: sample purification, enzymatic hydrolysis reaction, chromogenic reaction, standard curve drawing and the like. The method is simple in testing process and is rapid and easy to operate; complex special equipment and professional technicians are not needed; testing results are visual and clear, can be directly visually detected and judged, can be accurately quantified, are high in repeatability, and are not easily interfered by impurities; melamine in the sample can be effectively enriched; the high-flux measurement condition is met; and the demand of common consumers in China on simple detection of the melamine is satisfied.

Description

technical field [0001] The invention relates to the detection field, in particular to a colorimetric detection method for melamine based on an enzymatic amino hydrolysis reaction. Background technique [0002] Melamine (Melamine), structure as shown in the compound of formula I, is a kind of triazine nitrogen-containing heterocyclic organic compound, chemical formula is C 3 h 6 N 6 , commonly known as melamine, protein essence. [0003] [0004] Melamine is an important chemical raw material used to make plastics. However, it is toxic and is not allowed to be used as a food additive. When melamine is repeatedly ingested over a long period of time, it can cause severe kidney and bladder damage, sometimes fatal. However, due to its low price and high nitrogen content, melamine is illegally added to foods and feeds to falsely increase protein content, such as infant formula, milk, and other foods. With the country's attention to food safety issues and the melamine scan...

Claims

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Application Information

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IPC IPC(8): G01N21/78
Inventor 周佳海黄维雪张志民
Owner SHANGHAI INST OF ORGANIC CHEM CHINESE ACAD OF SCI
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