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Dynamic sandwich structure-based electrochemical sensor, and making method and application thereof

An electrochemical and sandwich technology, applied in the field of bioelectrochemistry, can solve the problems of insufficient sensitivity and accuracy, and achieve the effects of improved detection sensitivity, convenient analysis of precious samples, and simple operation.

Active Publication Date: 2016-08-31
NANJING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Despite these advantages, the use of electrochemical techniques to study protein interactions is still exploratory and often suffers from insufficient sensitivity and precision

Method used

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  • Dynamic sandwich structure-based electrochemical sensor, and making method and application thereof
  • Dynamic sandwich structure-based electrochemical sensor, and making method and application thereof
  • Dynamic sandwich structure-based electrochemical sensor, and making method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1: Reaction process for detection of goat anti-digoxigenin immunoglobulin by dynamic sandwich structure electrochemical sensor

[0038] Step 1: Fix the gold electrode with a diameter of 3 mm upside down, mix 98% concentrated sulfuric acid and hydrogen peroxide at a ratio of 3:1 to prepare a piranha solution, add 10 μL of piranha solution to completely cover the gold surface, and wait for 10 minutes Afterwards, rinse with a large amount of ultrapure water; gently polish the gold electrode with 1.0 μm alumina powder on silk cloth and 0.3 μm alumina powder on suede for 5 minutes each, so that the surface of the electrode is smooth to the mirror surface; rinse and polish with a large amount of ultrapure water The surface of the gold electrode was immersed in ethanol and ultrapure water successively, and ultrasonically cleaned for 5 minutes; the surface of the electrode was blown dry with nitrogen, and 10 μL of piranha solution was added dropwise on the surface of the...

Embodiment 2

[0041] Example 2: Detection of Antibody-Antibody Interaction Reaction Process by Dynamic Sandwich Structure Electrochemical Sensor

[0042] image 3 It is a schematic diagram of the dynamic sandwich structure electrochemical sensor for antibody-antibody interaction analysis. On the basis of Example 1, if rabbit anti-goat immunoglobulin (Ab2) that can interact with goat anti-digoxigenin immunoglobulin (Ab1) is further introduced into the detection system, the concentration is set at 0.01nM, 0.5nM , 1.5nM, 5nM, 8nM, 20nM, 200nM, 2000nM, due to the further increase in the volume of the formed "DNAzyme-goat antibody-rabbit anti-sheep antibody" complex structure, the DNAzyme cuts the signal chain more slowly ( Figure 4 ), within the same action time, the electrochemical signal is further enhanced, and by comparing the electrochemical signal of a single sheep antibody, the presence of rabbit anti-sheep antibody in the solution can be analyzed ( Figure 5 ).

Embodiment 3

[0043] Example 3: Sensitive detection of goat anti-digoxigenin immunoglobulin by an electrochemical sensor combined with a dynamic sandwich structure and gold nanoparticles Reaction process

[0044] Because the larger the volume of the adsorbate on the DNAzyme, the lower the ability to cut the signal chain on the gold electrode. If the DNAzyme is connected with gold nanoparticles to form a super-large-volume "DNAzyme-target-gold nanoparticle" composite structure, it will Greatly improve the analysis ability of the sensor.

[0045] Step 1, the gold nanoparticle solution (3nM) covered with 20nm citric acid was mixed with 100mM K 2 CO 3 The solution was adjusted to pH 9.0; 10 μL of 1 mg / mL rabbit anti-sheep antibody was added to 990 μL of the above gold nanoparticle solution, and incubated at room temperature for 2 h; centrifuged at 13,000 rpm for 15 min, the supernatant was discarded, and resuspended in 990 μL of PBS (pH 8.0). Repeat once to remove the antibody that is not bou...

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Abstract

The invention belongs to the technical field of bioelectrochemistry, and concretely relates to a dynamic sandwich structure-based electrochemical sensor, and a making method and an application thereof. The electrochemical sensor is composed of an electrode, a signal chain and a reaction system, the electrode adopts a gold electrode, the signal chain is a segment of single-stranded DNA, the 5' end of the DNA chain is modified with a mercapto group, the 3' end of the DNA chain is modified with methylene blue, the mercapto group and the surface of the gold electrode are self-assembled to form a gold-mercapto bond standing on the surface of the gold electrode, the reaction system contains DNAzyme and a necessary reaction buffer system, the DNAzyme can be complemented and paired to the signal chain and cuts the signal chain into two segments of DNA short strands, and the DNAzyme is marked with a target protein identification element, and can be used for quantitatively detecting proteins and interaction among the proteins. The electrochemical sensor is simple to operate, can be operated by operators without special training, and is suitable for being used in remote regions and resource-poor regions.

Description

1. Technical field [0001] The invention belongs to the technical field of bioelectrochemistry, and in particular relates to an electrochemical sensor based on a dynamic sandwich structure and its preparation and application. 2. Background technology [0002] There are approximately 650,000 protein interaction pairs in the human proteome. The interaction of these protein molecules controls most of the activities of cells. Deciphering the interaction network of these protein molecules can not only provide a deeper understanding of how these proteins are Function and ultimately affect cell activities, and help people design more specific drug molecules to treat specific diseases. Therefore, there is an inherent urgent need to develop sensitive and operator-friendly analytical methods to detect these protein-protein interaction events in the field of basic biological research. [0003] Currently, a variety of methods for studying protein-protein interactions have been developed...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N27/26
CPCG01N27/26G01N33/68
Inventor 李根喜李超马洁桦胡晓璐
Owner NANJING UNIV
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