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A biomarker detection method using electrochemiluminescence imaging recognition technology

A technology of biomarkers and luminescence imaging, which is applied in the fields of chemiluminescence/bioluminescence, biological testing, and analysis by making materials undergo chemical reactions, and can solve the problems of easy oxidation of electrodes

Active Publication Date: 2021-04-13
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The electrochemiluminescence imaging recognition system developed by the present invention is composed of an electrochemical module, a microscopic imaging system and an image acquisition system: the electrochemiluminescence module adopts a chip mode and is equipped with detachable printed electrodes to overcome the problems of easy oxidation and poor reproducibility of traditional electrodes. Problem; the microscopic imaging system uses a high-resolution microscope, and cooperates with a high-precision stepping stage and a chip fixing bracket to finally obtain stable and complete microscopic image information; the image acquisition system uses a high-sensitivity charge-coupled device (CCD) as Core components, and cooperate with temperature control and gain adjustment modules to achieve efficient image acquisition process

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  • A biomarker detection method using electrochemiluminescence imaging recognition technology
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  • A biomarker detection method using electrochemiluminescence imaging recognition technology

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Embodiment 1

[0068] Embodiment 1 Electrochemiluminescence imaging recognition method

[0069] 1. Overall technical route and process

[0070] The present invention aims to construct a single-molecule electrochemiluminescence imaging recognition system, and finally realize a biomarker detection method based on microRNA, telomerase and CEA protein. The technical route of the present invention is as figure 1 Shown:

[0071] 1.1 The present invention takes terpyridine ruthenium-lysine polymer as an entry point to synthesize linear and dendritic terpyridine ruthenium-lysine polymer molecules, thereby constructing a polymer amplification system, which is a single-molecule electrochemiluminescence imaging system Provide amplified electrochemiluminescence signal, and finally achieve the purpose of single molecule recognition. In order to further improve the convenience of the synthesis of linear terpyridine ruthenium-lysine polymers and dendritic terpyridine ruthenium-lysine polymers, an automa...

Embodiment 2

[0086] Example 2 Application of electrochemiluminescence imaging recognition method in detection of microRNA expression level

[0087] 1) microRNA expression level detection

[0088] Based on the electrochemiluminescence imaging technology amplified by polymers, the invention makes up for the problem of insufficient sensitivity of the non-amplified microRNA detection method. Its technical process mainly consists of the following steps, such as Figure 6 Shown:

[0089] ① Take 10 μM linear or dendritic terpyridine ruthenium-lysine polymers, add an equal amount of DNA recognition domain (concentration: 100 μM), and incubate overnight (12 hours) at 37°C to complete the signal amplification group That is, the linking of linear or dendritic terpyridine ruthenium-lysine polymers to the DNA recognition domain.

[0090] ② Take a 50K Dalton (Da) ultrafiltration tube, transfer the primary product obtained in step ① to the ultrafiltration tube, and refrigerate and centrifuge at 5000 r...

Embodiment 3

[0099] Example 3 Application of electrochemiluminescence imaging recognition method in detection of telomerase activity

[0100] The application of the electrochemiluminescence imaging recognition method in the detection of telomerase activity is based on the principle of electrochemiluminescence cascade amplification, and the electrochemiluminescence imaging recognition method is used as the signal giving mode (the schematic diagram is shown in Figure 7 shown), which mainly consists of the following steps:

[0101] Ⅰ The telomerase extraction step is treated with a commercial kit to obtain a telomerase extraction sample, and the telomerase extension treatment is used to obtain a telomerase extension product.

[0102] II The construction of the electrochemiluminescent signal amplification probe followed steps ① to ⑤ in Example 2.

[0103] III Take 1 μL of the electrochemiluminescent signal amplification probe obtained in step II, add 44.5 μL of ultrapure water, and add 2.5 μ...

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Abstract

The invention discloses a biomarker detection method using electrochemiluminescence imaging recognition technology. First, an electrochemiluminescence amplification probe is constructed, and the electrochemiluminescence amplification probe is added to an extracted sample, so that the biomarker and the electrochemiluminescence Connect signal amplification probes to obtain system 1; add capture probes to system 1 to connect biomarkers to capture probes to obtain system 2; add streptavidin magnetic beads to system 2, and vortex to mix thoroughly After homogenization, connect the biomarkers with streptavidin magnetic beads, then separate them with a magnetic separator, and wash with PBS 1X buffer; mix the products obtained in step 1 with ultrapure water, and then place them in an electrochemiluminescence imaging system Detect luminescent signal. The invention has the advantages of: 1) high signal acquisition sensitivity, visualization and quantitative statistical analysis; (2) good sample compatibility; (3) simple and fast operation, no need for sample amplification; The sample extraction process can be used for biomarker monitoring, which takes a short time, eliminates the amplification step, and detects quickly.

Description

technical field [0001] The invention belongs to the technical field of biomarker detection, in particular to a biomarker detection method based on a single-molecule electrochemiluminescent recognition system and its application. Background technique [0002] With the rapid development of life science technology, more and more biomarkers are playing an increasingly important role in related technical fields. Among them, major markers such as microRNA, telomerase, CEA protein and related coding RNA have become star molecules in life sciences, medicine and other disciplines. However, the existing detection technologies for these molecular markers have shortcomings such as inconsistent technical routes, poor compatibility of instruments and equipment, and inability to achieve collaboration between technical systems. Therefore, it is of great significance for the detection of existing biomarkers to develop a multi-functional detection platform that is versatile, highly sensitive...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/76G01N33/53
CPCG01N21/763G01N33/53
Inventor 黄曦廖玉辉赵钊艳谭青琴
Owner SUN YAT SEN UNIV