Method for electrochemically detecting activation level of STAT 3 protein through DNA probe ligand and application

A DNA probe and electrochemical technology, applied in the fields of molecular diagnosis and obstetric high-risk pregnancy detection, can solve problems such as affecting new signal amplification strategies, poor universality of detection systems, and difficulty in meeting life sciences.

Inactive Publication Date: 2017-09-08
孙丽洲
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

However, these classical methods relying on the antigen-antibody recognition mechanism have limitations to varying degrees, such as the highly complex chemical structure of the antibody, complicated preparation process and high price, cumbersome operation, time-consuming and need to contact radioactive substances, etc.
With the increasing interest in the specific detection of disease-related protein markers, in addition to immunoassays based on antigen-antibody binding, another type of protein biosensor based on chemically synthesized ligands of nucleic acid aptamers has also received great attention. The two complement each other, but the detection of the activation level of protein markers still faces new challenges in terms of sensitivity, selectivity, and response s

Method used

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  • Method for electrochemically detecting activation level of STAT 3 protein through DNA probe ligand and application
  • Method for electrochemically detecting activation level of STAT 3 protein through DNA probe ligand and application
  • Method for electrochemically detecting activation level of STAT 3 protein through DNA probe ligand and application

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Experimental program
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Effect test

Embodiment 1

[0059] AC impedance is an effective means to study the interface characteristics of surface-modified electrodes. In order to confirm the modification of the electrode surface, the change of the surface state of the gold electrode was first studied by EIS technique at various stages of detection. The AC impedance plot includes a semicircle in the high-frequency region associated with the interfacial electron transfer process, and a linear portion in the low-frequency region associated with the bulk solution diffusion process to the electrode surface. The increase of the diameter of the semicircle reflects the increase of the charge transfer resistance on the electrode surface.

[0060] figure 1 It clearly reflects the AC impedance on the surface of the gold electrode in different reaction stages. like figure 1 As shown in a, the impedance diagram of the bare gold electrode is almost a straight line, indicating that the transfer of electrons on the electrode surface has not b...

Embodiment 2

[0062] The gold electrode modified with the double-stranded DNA sequence of the capture probe was reacted with 100nM activated STAT 3 protein, 100nM non-activated STAT 3 protein, and 10mM TBS buffer (pH 7.4) respectively to investigate the effect of the present invention on the activated STAT 3 protein specificity.

[0063] Figure 4 In this study, when the functionalized gold electrode reacted with the activated STAT 3 protein through a series of experimental steps and finally introduced the graphene catalytic beacon, the catalyzed aniline oxidation reaction produced a large amount of 2,3- Diaminophenazine, resulting in a very pronounced SWV peak at -0.42mV. However, when the activated protein was replaced with the same concentration of non-activated STAT 3 protein or TBS buffer was used as a blank control to react with the electrode, since there was no protein binding on the electrode surface, all DNA was degraded, and ultimately no available Zr 4+ The identified phosphory...

Embodiment 3

[0065] Optimization of protein incubation time with capture probes. Considering that being able to capture the activated STAT 3 protein from the liquid phase and successfully immobilize it to the electrode interface is an important basis for the whole process, so this example sets the incubation time between the STAT 3 protein and the DNA capture probe on the electrode optimized ( image 3 ). The experimental results show that the peak current value of SWV reaches saturation at 120 minutes, which is enough to ensure that the activated STAT 3 can be recognized and bound by the DNA sequence of the capture probe, thereby being immobilized on the surface of the gold electrode. The incubation time for one step was chosen to be 120 minutes.

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Abstract

The invention discloses a method for electromagnetically detecting the activation level of an STAT 3 protein through a DNA probe ligand and application. By use of STAT3 which is an important transcription factor and dynamic reversible and controllable mutual action between two natural ligands which are DNA and polypeptide, a DNA probe capable of specifically identifying a protein is designed; the polypeptide is used for controlling reversible combination and dissociation between the target protein STAT3 and the DNA probe; by stable coordination interaction between Zr<4+> and a phosphate group and a phosphorylated nano-graphene composite beacon, catalytic signal amplification is realized. According to the method, by introduction of the phosphorylated nano-graphene composite beacon serving as a signal amplification strategy, the phosphorylated nano-graphene composite beacon is stable and difficult to inactivate, thus finally realizing sensitive, efficient and stable detection for the activated STAT3 protein.

Description

technical field [0001] The invention belongs to the field of molecular diagnosis and high-risk pregnancy detection in obstetrics, and relates to a method and application for electrochemically detecting STAT 3 protein activation level by using a DNA probe ligand. Background technique [0002] At present, the detection and analysis of protein activation level mainly rely on traditional immunological methods such as Western Blotting and ELISA; the detection of STAT 3 protein activation level is also mainly through these two methods. However, these classical methods relying on antigen-antibody recognition mechanisms have limitations to varying degrees, such as the highly complex chemical structure of antibodies, complicated preparation processes and high prices, cumbersome operations, time-consuming, and the need to contact radioactive substances. With the increasing interest in the specific detection of disease-related protein markers, in addition to immunoassays based on antig...

Claims

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Application Information

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IPC IPC(8): G01N27/327G01N27/416G01N27/48
CPCG01N27/3278G01N27/416G01N27/48
Inventor 孙丽洲殷婷婷黄欢
Owner 孙丽洲
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