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Colorimetric analysis method for detecting kanamycin based on aptamer modified magnetic bead and gold nanoparticle mimic enzyme activity

A technology of gold nanoparticles and kanamycin, applied in the field of analytical chemistry, can solve the problems of large error in results, expensive equipment, complicated operation, etc., and achieve the effects of high specificity, good signal amplification, and easy preparation

Inactive Publication Date: 2017-10-10
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The microbiological detection method is widely used, and its advantage is that it is low in cost and can be operated by general laboratories, but the measurement time is long, the result error is large, the operation is complicated, and it cannot meet the needs of specificity and quantitative detection.
The instrumental inspection method uses the structure and physical and chemical properties of kanamycin for separation and qualitative and quantitative determination. It has good accuracy, but the equipment is often expensive, and it also has high requirements for experimenters and high requirements for sample pretreatment. Analysis Time-consuming and difficult to promote

Method used

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  • Colorimetric analysis method for detecting kanamycin based on aptamer modified magnetic bead and gold nanoparticle mimic enzyme activity
  • Colorimetric analysis method for detecting kanamycin based on aptamer modified magnetic bead and gold nanoparticle mimic enzyme activity
  • Colorimetric analysis method for detecting kanamycin based on aptamer modified magnetic bead and gold nanoparticle mimic enzyme activity

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Experimental program
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Effect test

Embodiment 1

[0024] Specific steps are as follows:

[0025] (1) Preparation of gold nanoparticle-mimicking enzyme AuNPs: prepared by using tyrosine as a reducing agent and capture agent. Gold nanoparticle-mimicking enzyme: 300 mL ultrapure Boil the aqueous solution for 3-5 minutes, immediately add 1.02 mL of chloroauric acid solution with a mass volume ratio of 1%-3%, and keep warm in the boiling water bath for 5-10 minutes; continue to boil in the boiling water bath until the solution volume is reduced to 30 mL, Obtain gold nanoparticle solutions of different particle sizes; dialyze the above gold nanoparticle solution in advance in a dialysis bag with a molecular weight cut-off of 12 kDa treated in boiling water to remove excess KOH, metal ions and tyrosine, and obtain gold nanoparticle simulation Enzyme AuNPs, stored in a 4°C refrigerator for later use;

[0026] (2) Preparation of functionalized gold nanoparticles (AuNPs-cDNA): All reagent bottles and Ep tubes used need to be soaked in...

Embodiment 2

[0033] The detection of embodiment 2 kanamycin standard solution

[0034] Different concentrations of kanamycin solutions were selected and added to the reaction system to form a specific structure with the kanamycin aptamer sequence through a displacement reaction to replace AuNPs-cDNA from the magnetic beads. After magnetic separation, the free AuNPs were absorbed, transferred to another Ep tube, and the catalytic reaction substrates TMB and H 2 o 2 After a period of reaction, 2 M H 2 SO 4 Terminate the reaction, centrifuge, test its UV-vis spectral curve in a spectrophotometer, and record the absorbance at 450 nm, according to the relationship between the concentration of kanamycin in the sample solution and the absorbance at 450 nm, Draw the corresponding relationship curve and linear range curve ( figure 2 , image 3 ). In the range of 5-100 nM kanamycin standard solution concentration, there is a good linear relationship between the light absorbance value at 450 n...

Embodiment 3

[0035] Example 3 Detection of Kanamycin Residues in Honey Samples

[0036] Here, the method of artificially polluting honey is prepared by adding standard concentration of kanamycin to honey to obtain honey samples containing 0-1000nM kanamycin residue.

[0037] The artificially polluted kanamycin in acacia honey was detected according to the steps of standard substance detection in Example 2.

[0038] get as Figure 4 UV-vis spectral curves under different kanamycin concentrations shown, and Figure 5 The linear relationship between the light absorbance value and the concentration of kanamycin in the honey sample is shown.

[0039] In the range of 2-1000 nM, the light absorption value is positively correlated with the concentration of kanamycin (nM), and in the range of 5-100 nM, it shows a good linear relationship. The linear equation is y=0.0018x+0.1082, R 2 =0.9877, the detection limit was 18.3 nM. The meaning of letters in the formula is the same as in Example 1. Th...

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Abstract

The invention discloses a colorimetric analysis method for detecting kanamycin based on aptamer-modified magnetic beads and gold nanoparticles to simulate enzyme activity, and belongs to the technical field of analytical chemistry. In the present invention, the gold nanoparticles synthesized by tyrosine reduction of chloroauric acid simulate the peroxidase-like activity inherent in the enzyme AuNPs, and the gold nanoparticles modified by kanamycin-specific aptamers modify their complementary single-stranded cDNA The capture of nanoparticles, and the displacement of gold nanoparticles by the combination of kanamycin and aptamer, the peroxidation-like peroxidation of gold nanoparticles AuNPs contained in the supernatant after magnetic separation is related to the concentration of kanamycin The enzyme activity catalyzes the color reaction between the substrate tetramethylbenzidine (TMB) and H2O2, realizes the visual detection of kanamycin, and uses the linear relationship between the absorption value at 450 nm and the concentration of kanamycin to realize the detection of kanamycin. Colorimetric quantitative analysis of kanamycin. This method has the advantages of high sensitivity and good specificity, and is suitable for the quantitative analysis of kanamycin residues in food samples such as honey.

Description

technical field [0001] The invention relates to a colorimetric analysis method for detecting kanamycin based on aptamer-modified magnetic beads and gold nanoparticles to simulate enzyme activity, and belongs to the technical field of analytical chemistry. Background technique [0002] Kanamycin is a broad-spectrum aminoglycoside antibiotic with the molecular formula C 18 h 38 N 4 o 15 S, molecular weight 582.58. Kanamycin is mainly extracted from the fermentation broth of Streptomyces griseus or Micromonas or semi-synthetically extracted from natural products. As a water-soluble antibiotic, Kanamycin Sulfate is effective against many Gram-negative bacteria including Salmonella, Tuberculosis and some Gram-positive bacteria. It is widely used as a medicine for humans and animals with excellent effect. In agriculture, animal husbandry, and aquaculture, kanamycin can not only treat various infectious diseases, but also promote the growth of livestock and poultry and improv...

Claims

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Application Information

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IPC IPC(8): G01N33/543G01N21/31
CPCG01N33/54346G01N21/31
Inventor 周楠迪王春帅姚斌斌田亚平
Owner JIANGNAN UNIV
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