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Temperature controllable electrochemical mercury ion sensor and preparation method thereof

An electrochemical and sensor technology, applied in the field of nucleic acid detection and biological analysis, can solve the problems of increasing the electrode response signal, denaturing the molecular structure of the enzyme, shortening the sensor time, etc., to increase the electrode response signal, enhance the activity, and improve the detection sensitivity Effect

Active Publication Date: 2017-10-03
FUZHOU UNIVERSITY
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  • Abstract
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Problems solved by technology

[0004] Exonuclease III is a biological macromolecule that is very sensitive to temperature changes. When the temperature is lower than the optimum temperature, the activity of the enzyme is poor, but if the temperature is too high, the molecular structure of the enzyme will be irreversibly denatured and the enzyme will be inactivated.
Most of the amperometric biosensors reported in the past control the overall temperature change of the experimental system, and the required devices are complicated and difficult to operate; building a biosensor on the surface of the thermode can only change the temperature of the electrode surface to keep the enzyme in the optimal condition without the overall solution. Heating enhances the convection of the solution, improves the mass transfer rate, shortens the time for the sensor to reach the steady-state current, and increases the electrode response signal

Method used

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  • Temperature controllable electrochemical mercury ion sensor and preparation method thereof
  • Temperature controllable electrochemical mercury ion sensor and preparation method thereof
  • Temperature controllable electrochemical mercury ion sensor and preparation method thereof

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Embodiment 1

[0038] A Temperature-Controllable Electrochemical Hg-Based Amplification of Exonuclease III Targeted Cycling Signals 2+ The preparation method of the sensor, such as figure 1 shown, including the following steps:

[0039] (1) Design a signal probe P1 that is labeled with ferrocene (Fc) near the 5', and the complementary presence of the signal probe P1 and the DNA auxiliary probe P2 can recognize Hg 2+ The T-T mismatch structure, in Hg 2+ In the presence, P2 hybridizes with P1 to form a T-Hg 2 +A double-stranded structure with a 3' blunt end of the -T structure. Induces exonuclease III to digest P1, Hg 2+ Released for recycling; thiol at the 5' end of the P1 chain;

[0040] Wherein, the nucleotide sequence of signal probe P1 is: 5'-SH-(CH 2 ) 6 -CCCCA T(Fc)CGCC ACCAG CTTCT-3',

[0041] The nucleotide sequence of the auxiliary probe P2 is: 5'-TGTAG CTGGT GGCGA TCCCA C-3';

[0042] (2) Gold disk thermode on suede with 0.05 mm Al 2 o 3 Polished to a mirror surface, ul...

Embodiment 2

[0046] Reaction time optimization experiment:

[0047] The enzyme digestion cycle reaction time in step (4) of Example 1 was changed to 0, 15, 30, 45, 60, 75, 90, 105, and 120 min. respectively at different electrode temperatures (0 ºC, 25 ºC, 40 ºC) The experiment was carried out, and other reaction conditions were the same as in Example 1; the gold disc thermode modified with the sulfhydrylation signal probe P1 obtained in step (3) of Example 1 was subjected to SWV detection in 10 mM tris-HCl detection solution to obtain the Fc Oxidation peak current I 0(Fc) ; The electrochemical biosensor finally obtained in the embodiment 1 step (4) is detected with SWV in 10 mM tris-HCl detection solution, and the oxidation peak current I of the obtained Fc is Fc with the obtained I 0(Fc) Take the absolute value of the difference: |ΔI Fc |=|I Fc -I 0(Fc) | with |ΔI Fc |Plotted against temperature, such as figure 2 As shown, at the same electrode temperature, it can be seen that th...

Embodiment 3

[0049] Reaction temperature optimization experiment:

[0050] Only change the reaction temperature in step (4) of Example 1, where a is the control group, and the reaction temperatures of b-f are 0, 10, 20, 24, 30, 35, 40 ºC in sequence, and the experiments are carried out separately, and other reaction conditions are implemented at the same time Example 1: The gold disc thermode modified by the signal probe P1 obtained in step (3) of Example 1 was subjected to SWV detection in 10 mM tris-HCl detection solution, and the oxidation peak current I of Fc was obtained 0(Fc) ; The electrochemical biosensor finally obtained in the embodiment 1 step (4) is detected with SWV in 10 mM tris-HCl detection solution, and the oxidation peak current of the obtained Fc is compared with the obtained I 0(Fc) Take the absolute value of the difference: |ΔI Fc |=|I Fc -I 0(Fc) | with |ΔI Fc |Plotted against temperature, such as image 3 As shown, it can be seen that with the increase of temper...

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Abstract

The present invention discloses a temperature-controllable electrochemical Hg<2+> sensor based on exonuclease (Exo III) target circulating signal amplification, and a preparation method thereof. The sensor comprises a gold plate thermal electrode, a signal probe P1, an auxiliary probe P2 and exonuclease III, the T-T mismatching structure capable of identifying Hg<2+> can be produced through the complementation of P2 and P1, the T-T mismatching structure and Hg<2+> for a double-stranded structure having a T-Hg<2+>-T structure and having a 3' blunt end, the exonuclease III is induced to digest the P1, the amount of the P1 on the surface of the electrode is decreased so as to decrease the Fc chemical signal, and the decreasing degree of the Fc chemical signal on the electrode surface and the Hg<2+> concentration form linear relationship, such that the high sensitivity detection on the Hg<2+> can be achieved.

Description

technical field [0001] The invention belongs to the technical field of biological analysis, in particular to a temperature-controllable electrochemical mercury ion sensor and a preparation method thereof, which are applied in the field of nucleic acid detection. Background technique [0002] heavy metal ion Hg 2+ Due to their relatively stable properties, they are difficult to be biodegraded in the natural environment and have strong biological toxicity. They will eventually remain in the human body through the enrichment of the food chain and destroy various physiological functions of the human body. Human tissues and organs even pose a serious threat to life. Therefore, the establishment of a rapid and reliable detection method for mercury ions is of great significance in environmental monitoring and food safety testing. [0003] Traditional heavy metal ion detection methods mainly rely on large-scale professional instruments. The main disadvantages of these methods are ...

Claims

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Application Information

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IPC IPC(8): G01N27/327G01N27/30
CPCG01N27/30G01N27/3271
Inventor 吴韶华王芳芳孙建军张标米真真
Owner FUZHOU UNIVERSITY
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