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Aptamer electrochemical sensor used for kanamycin A detection and production and application methods of aptamer electrochemical sensor

A technology of nucleic acid aptamer and kanamycin, which is applied in the field of biological analysis, can solve problems such as the influence of test results and the need to verify the tolerance, and achieve the effect of simple design and strong versatility

Active Publication Date: 2015-09-02
CAPITAL NORMAL UNIVERSITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, due to the limitations of the electrochemical impedance detection method itself, non-specific adsorption will affect the detection results, and the tolerance of this method to other target molecules needs to be verified.

Method used

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  • Aptamer electrochemical sensor used for kanamycin A detection and production and application methods of aptamer electrochemical sensor
  • Aptamer electrochemical sensor used for kanamycin A detection and production and application methods of aptamer electrochemical sensor
  • Aptamer electrochemical sensor used for kanamycin A detection and production and application methods of aptamer electrochemical sensor

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Experimental program
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Effect test

Embodiment 1

[0034] Embodiment 1: the two-step method preparation of SD-EAB A and B that is used for kanamycin A detection.

[0035] SD-EAB A( figure 1 A ) preparation: 1 μM capture probe 1 (A-SH) and 100 μM TCEP in buffer A (10 mM phosphate buffer, 1 M NaCl, 5 mM MgCl 2 , pH 7.0), mix well, and let stand for reduction for 1 hour. Immerse the clean electrode in the reducing solution, place it at 37°C, and react overnight. Wash with buffer B (10mM phosphate buffer, 1M NaCl, pH 7.0) three times, and then put into 2mM MCH for blocking at 37°C for 1 hour. After washing with buffer B three times, it was immersed in buffer A containing 0.5 μM signal probe 2 (C-Fc) and incubated at 37° C. for 2 hours, and washed with buffer B three times. The prepared sensor was stored in the hybridization solution at 4°C for future use.

[0036] SD-EAB B( figure 1 B ) preparation: the above capture probe 1 (A-SH) was replaced with capture probe 3 (C-SH), the above signal probe 2 (C-Fc) was replaced with s...

Embodiment 2

[0037] Embodiment 2: Utilize SD-EAB A and SD-EAB B to detect the kanamycin of different concentrations respectively.

[0038] Use electrochemical equipment to scan the two sensors with square wave voltammetry, and measure the oxidation peak corresponding to ferrocene near 0.2V. Detection of Namycin A. Kanamycin A was detected by SD-EAB A and SD-EAB B, respectively. get results like figure 2 As shown, the kinetic interval of the SD-EAB A sensor in the present invention is 1 nM to 10 mM, which is 2-5 orders of magnitude wider than that of the kanamycin A sensor reported in the prior art. The logarithm of the concentration of Kanamycin A has a good linear relationship with the corresponding current change in the range of 1nM to 100μM, the correlation coefficient is 0.996, and the detection limit is 1nM, which is better than other kanamycins based on nucleic acid aptamers reported. The A sensor is 5-10 times more sensitive, comparable to immunological and spectroscopic methods...

Embodiment 3

[0039] Embodiment 3: SD-EABA among the present invention is carried out target selectivity assay.

[0040] Antibiotics with similar or completely different chemical structures to kanamycin A were tested with SD-EAB A in the same manner. SD-EAB A has excellent selectivity to the structural analog of kanamycin A (kanamycin B) and other types of antibiotics including ampicillin, sulfidexoxine and tetracycline. When the concentration of the antibiotic tested was 100 μM, the corresponding current signal decreases were: kanamycin A 51.2%, sulfidesoxine 0±9.4%, tetracycline 2.2±6.8%. In the presence of kanamycin B and ampicillin, an increase in the current signal was detected, corresponding to an increase in the current signal of 9.8±1.0% and 6.6±1.6%, respectively. Even at a high concentration (1 mM), SD-EAB still has a high degree of specific recognition for kanamycin A.

[0041] When SD-EAB A interacts with kanamycin A and its structural analogue kanamycin B, it shows completely...

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Abstract

The invention relates to a kanamycin A aptamer electrochemical sensor based on signal probe chain substitution reaction and production and application methods of the kanamycin A aptamer electrochemical sensor. SD-EAB consists of a sulfhydryl-modified capture probe (an aptamer or a short complementary chain) and a signal probe (a short complementary chain or an aptamer) which is complementary to and is hybridized with the capture probe and has an oxidation-reduction mark. When kanamycin A exists, the signal probe is substituted and released from the surface of an electrode, so as to result in decreasing of current, and a decrease value of current is proportional to a logarithm of the concentration of the kanamycin A. The signal transduction of the kanamycin A aptamer electrochemical sensor provided by the invention is only caused by affine competition among target molecules, the short complementary chain and the aptamer, and is not associated with a conformation state of the aptamer, so that the generality of the aptamer is greatly improved. The SD-EAB has the advantages of high sensitivity, good specificity, wide dynamic interval and strong anti-jamming capability, without additional reagents.

Description

technical field [0001] The invention relates to a nucleic acid aptamer electrochemical sensor (SD-EAB) based on the substitution of a signal probe chain for kanamycin A detection, a production method and an application method thereof, and belongs to the technical field of bioanalysis. Background technique [0002] Nucleic acid aptamer (Aptamer) is a DNA (deoxyribose nucleic acid) or RNA (ribonucleic acid) sequence obtained through in vitro screening, which can bind to a variety of target substances with high specificity and high selectivity. Compared with antibodies, it has the ability to artificially Synthesis, good stability, convenient chemical modification and engineering design and other advantages, so it has a good application prospect in the field of biosensors. [0003] Antibiotics play an extremely important role in the treatment of infectious diseases, but due to serious abuse in recent years, the problem of antibiotic residues in animal foods has become prominent....

Claims

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Application Information

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IPC IPC(8): G01N27/327G01N27/48
Inventor 娄新徽刘然何苗赵俊才康倩唐云飞
Owner CAPITAL NORMAL UNIVERSITY
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