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Biomarker detection method using electrochemical luminescence imaging identification technology

A technology of biomarkers and luminescent 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: 2018-03-06
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

Method used

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  • Biomarker detection method using electrochemical luminescence imaging identification technology
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  • Biomarker detection method using electrochemical luminescence imaging identification 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 Image 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 rp...

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 processed with a commercial kit to obtain a telomerase extraction sample.

[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 μL of phosphate buffer saline (PBS, 20X), and vortex.

[0104] IV Add 1 μL of telomeras...

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Abstract

The invention discloses a biomarker detection method using electrochemical luminescence imaging identification technology. The biomarker detection method comprises the following steps: firstly, constructing an electrochemical luminescence amplification probe, adding the electrochemical luminescence amplification probe into an extracted sample to connect a biomarker with the electrochemical luminescence amplification probe, so as to obtain a system 1; adding a capture probe into the system 1 to connect the biomarker with the capture probe, so as to obtain a system 2; adding streptavidin magnetic balls into the system 2 for full vortex uniform mixing to connect the biomarker with the streptavidin magnetic balls, separating the streptavidin magnetic balls with a magnetic separator, flushing the streptavidin magnetic balls with a PBS (Phosphate Buffer Solution) 1X, uniformly mixing the product obtained in the step 1 with superpure water, and putting the mixture into an electrochemical luminescence imaging system to detect a luminescence signal. The biomarker detection method has the advantages that (1) the signal acquisition sensitivity is high and vivid, and statistic analysis can bequantified; (2) the sample is high in compatibility; (3) the operation is simple and quick, and no sample amplification is needed. The detection process of the biomarker detection method is simple; bya simple sample extraction process, the monitoring of the biomarker can be carried out; the time is short, the amplification step is eliminated, and the detection is fast.

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 Applications(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