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Nucleic acid detection method based on polymer electrochemiluminescence signal amplification technology

A technology of luminescent signal and detection method, which is applied in the direction of biochemical equipment and methods, measurement/inspection of microorganisms, etc., can solve the problems of not achieving ideal amplification effect, unstable labeling process, complicated reaction process, etc., and achieves convenience for modification and storage , easy to save, easy to operate

Active Publication Date: 2015-06-03
SOUTH CHINA NORMAL UNIVERSITY
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Problems solved by technology

[0004] However, there are some deficiencies in the nano-gold amplification technology: (1) The nano-gold amplification technology uses sulfhydryl groups to form sulfur-gold bonds on the surface of gold spheres, and the reaction process is complicated and time-consuming; (2) the nano-gold amplification technology requires nano The gold surface is often marked with multiple DNA probes, which is uncontrollable, resulting in multiple DNA probes being marked on a single nano-gold ball. Therefore, the ideal amplification effect cannot be achieved; (3) Nano-gold amplification technology often occurs during the labeling process. Nanogold aggregates, the labeling process is unstable and directly related to the quality of nanogold synthesis
Therefore, the process requires high technical

Method used

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  • Nucleic acid detection method based on polymer electrochemiluminescence signal amplification technology
  • Nucleic acid detection method based on polymer electrochemiluminescence signal amplification technology
  • Nucleic acid detection method based on polymer electrochemiluminescence signal amplification technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1 3

[0063] Synthesis and activation of embodiment 1 terpyridine ruthenium

[0064] (1) The synthesis process of terpyridine ruthenium is as follows:

[0065] ① Electronic balance weighs: dichlorobis(2,2'-bipyridine) ruthenium (Ru(bpy) 2 Cl 2 ) (CAS: 15746-57-3) 0.05g, NaHCO 3 0.05g, 0.0375g of 4-carboxylic acid-2,2'-bipyridine in a 50mL round bottom flask.

[0066] ② Add 10 mL of 80% methanol aqueous solution (MeOH), and heat the mixture under reflux in a silicone oil bath at 80°C for 10 h.

[0067] ③ The resulting solution was cooled in an ice bath (ice-water mixture) for 2 hours.

[0068] ④ Use 1mol / L of H 2 SO 4 Adjust the pH to 4.4 (calibrated with precision pH paper), and a precipitate forms.

[0069] ⑤ Filter the formed precipitate with filter paper and collect the filtrate.

[0070] ⑥Add 3.125mL NaPF to the filtrate obtained in ⑤ 6 (CAS: 21324-39-0) solution (2.5gNaPF 6 dissolved in 12.5mL of water), stirred and placed in the refrigerator at 4°C to evaporate the f...

Embodiment 2

[0079] The preparation of embodiment 2 polylysine-terpyridine ruthenium complex:

[0080] ① Take a tube of polylysine solid powder (5mg) and centrifuge at 12000r / min for 5min, so that the polylysine solid powder stuck on the tube wall falls to the bottom of the tube.

[0081] ② Gently open the cap of the tube, and quickly add 75 μL of 0.1M sodium borate (adjust the pH value to 8.5 with dilute hydrochloric acid), and immediately cap the tube.

[0082] ③ Fully oscillate up and down for 5 minutes, then centrifuge at 12000r / min for 5 minutes to collect, so that the liquid sticking to the tube wall falls to the bottom of the tube.

[0083] ④ Add 30 μL of the activated ruthenium terpyridine obtained in Example 1.

[0084] ⑤ Wrap it with black tape, place it on a centrifuge tube rack and incubate at room temperature in the dark for 12 hours.

[0085] ⑥Use an ultrafiltration tube (100KDa) to separate and remove small molecule compounds in the system.

[0086] ⑦ Centrifuge at 12000r...

Embodiment 3

[0095] Example 3 Magnetic capture detection target DNA sequence:

[0096] In this part, based on the traditional magnetic capture and "sandwich" model, a nucleic acid detection method using polylysine-terpyridine ruthenium as the signal amplification group is constructed.

[0097] (I) Dissolve the biotin probe and target sequence in water with a final concentration of 10 μM.

[0098] (II) Construct a magnetic capture nucleic acid detection system: the total volume of the system is 100 μL, the final concentration of biotin probe and polylysine is 100 nM, the final concentration of PBS buffer is 1×, add water to make up to 100 μL.

[0099] (Ⅲ) Signal detection: put the product of step (Ⅱ) into a Roche Elecsys 2010 electrochemiluminescence automatic immunoassay analyzer to detect the electrochemiluminescence signal, and record the data. Experimental results such as Figure 4 shown. The results showed that the electrochemiluminescence signal was significantly enhanced by using ...

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Abstract

The invention discloses a nucleic acid detection method based on the polymer electrochemiluminescence signal amplification technology. The method comprises the following steps: synthesizing and activating ruthenium terpyridine, preparing a polylysine-ruthenium terpyridine compound, marking the polylysine-ruthenium terpyridine compound with a DNA (deoxyribonucleic acid) probe, and magnetically capturing and detecting a target DNA sequence. According to the method, the polymer electrochemiluminescence signal amplification technology is applied to nucleic acid detection, and an organic polymer such as polylysine is good in physical and chemical properties and can be applied to the field of electrochemiluminescence signal amplification; polylysine is adopted as a connecting framework for ruthenium terpyridine signal amplification, and the system is stable, hard to aggregate and easy to store; the polylysine-ruthenium terpyridine compound marked with the DNA probe is stable in properties and convenient to modify and store. The method is easy in operation, quick, stable and controllable, is suitable for nucleic acid detection and can serve as an antibody labeling method in immunoassay to improve the sensitivity.

Description

technical field [0001] The invention belongs to the technical field of nucleic acid electrochemical detection, in particular to a nucleic acid detection method based on polymer electrochemical luminescence signal amplification. Background technique [0002] Nucleic acid electrochemical detection technology is an important detection method, which is widely used in the fields of immunodiagnosis, pathogenic microorganism screening and environmental monitoring. The method is based on molecular biology technology (often using nucleic acid amplification technology) and supported by the principle of electrochemiluminescence, aiming to construct a highly sensitive, fast and reliable detection method. Thanks to the superiority of the principle of electrochemiluminescence, this method has the characteristics of wide detection range, high sensitivity, controllable reaction system and high signal-to-noise ratio. [0003] However, the nucleic acid electrochemical detection technology us...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6816C12Q2563/143C12Q2525/10C12Q2565/401
Inventor 邢达周小明廖玉辉
Owner SOUTH CHINA NORMAL UNIVERSITY
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