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Structure-switching aptamer based highly-sensitive unmarked fluorescent detection method

A fluorescence detection and aptamer technology, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc., to achieve the effects of high detection specificity, resistance to interference from analogues and interferents, and low cost

Inactive Publication Date: 2012-09-19
CAPITAL NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

(Wang, X.L.; Li, F.; Su, Y.H.; Sun, X.; Li, X.B.; Schluesener, H.J.; Tang, F.; Xu, S.Q., Anal. Chem. 2004, 76, 5605-5610.) However This approach is only applicable to structure-switched aptamers that form tetramers

Method used

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  • Structure-switching aptamer based highly-sensitive unmarked fluorescent detection method
  • Structure-switching aptamer based highly-sensitive unmarked fluorescent detection method
  • Structure-switching aptamer based highly-sensitive unmarked fluorescent detection method

Examples

Experimental program
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Effect test

Embodiment 1

[0033] Example 1 uses probe 1 to detect different concentrations of K +

[0034] 50mM Tris-HCl, 2mM MgCl 2 , pH 8.3 Probe 1 was diluted to a concentration of 10 μM, heated at 90 °C for 5 min, and then slowly cooled to room temperature. Simultaneously with 50mM Tris-HCl, 2mM MgCl 2 , pH 8.3 diluted to prepare different concentrations of K + . Take 10 μL of probe 1 solution and 969 μL of different concentrations of K + The solution was mixed evenly in a centrifuge tube, and reacted for 12 hours at room temperature. Add 5U Exo I respectively, react at 37°C for 40min, and then heat at 80°C for 15min to stop the enzyme cleavage reaction. Then add 20μL of 50mM Tris-HCl, 2mM MgCl 2 , pH 8.3 diluted 10X SYBR Gold solution was mixed evenly, and stained for 20min. Scan and analyze the results with an atomic fluorescence spectrometer F-4500 from Shanghai Hitachi figure 2 (A).

[0035] The results show that with K + As the concentration increases, the fluorescence intensity in...

Embodiment 2

[0036] Example 2. Containing Na + Detection of K in the buffer + , examine Na + to K + detection interference.

[0037] 50mM Tris-HCl, 2mM MgCl 2 , 1 mM NaCl, pH 8.3 Probe 1 was diluted to a concentration of 10 μM, heated at 90 °C for 5 min, and then slowly cooled to room temperature. Simultaneously with 50 mM Tris-HCl, 2mM MgCl 2 , 1mM NaCl, pH 8.3 by diluting to prepare different concentrations of K + . Take 10 μL of probe 1 solution and 969 μL of different concentrations of K + The solution was mixed evenly in a centrifuge tube, and reacted for 12 hours at room temperature. Add 5U Exo I respectively, react at 37°C for 40min, and then heat at 80°C for 15min to stop the enzyme cleavage reaction. Then add 20μL of 50mM Tris-HCl, 2mM MgCl 2 , 1mM NaCl, pH 8.3 diluted 10X SYBR Gold solution was mixed evenly, and stained for 20min. Scan and analyze the results with an atomic fluorescence spectrometer F-4500 from Shanghai Hitachi figure 2 Solid line in (B).

[0038] 5...

Embodiment 3

[0040] Embodiment 3: investigate this fluorescence method to K + Specificity of detection.

[0041] 50mM Tris-HCl, 2mM MgCl 2 , pH 8.3 Probe 1 was diluted to a concentration of 10 μM, heated at 90 °C for 5 min, and then slowly cooled to room temperature. Simultaneously with 50mM Tris-HCl, 2mM MgCl 2 , pH 8.3 by diluting to prepare 1 mM of the different ions. Take 10 μL of probe 1 solution and 969 μL of solutions of different ions and mix them evenly in a centrifuge tube, and react at room temperature for 12 hours. Add 5U Exo I respectively, react at 37°C for 40min, and then heat at 80°C for 15min to stop the enzyme cleavage reaction. Then add 20μL of 50mM Tris-HCl, 2mM MgCl 2 , pH 8.3 diluted 10X SYBR Gold solution was mixed evenly, and stained for 20min. The atomic fluorescence photometer F-4500 of Shanghai Hitachi company was used to scan and analyze the results as follows: image 3 (A)-(B) shown.

[0042] The results show that the common cations on K + Detection in...

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Abstract

The invention relates to a method for unmarked fluorescent detection of ions, protein and micromolecules. According to the method, a target in presence can be combined with aptamers to enable the aptamers to be folded into secondary structures, and the secondary structures are more difficult to degrade by nuclease than the unfolded aptamers, the quantity of the remaining aptamers after enzyme digestion is in direct proportion to the concentration of the target, the remaining aptamers can be quantitated by nucleic acid dye SYBR Gold, and the fluorescent dye can only used for dyeing nucleic acid chains and has no dyeing effect to a product obtained after the enzyme digestion. A fluophotometer is used for scanning samples and analyzing fluorescence intensity, and detection of the target is realized by means of change of the fluorescent signals. The higher the concentration of the target in the sample is, the stronger the fluorescent signals are. The method can be used for detecting various targets such as K+, thrombase and cocaine and high-throughput and multipath detection, and can realize low-cost, highly-sensitive, accurate and rapid detection of the targets.

Description

technical field [0001] The invention relates to the technical field of biological analysis and detection, and more specifically relates to a label-free fluorescence detection method based on a structural switch aptamer, SYBR Gold and Exo I and its application. Background technique [0002] Nucleic acid aptamers are single-stranded DNA or RNA with a specific secondary structure, which can be obtained by in vitro screening. There are many types of targets, such as small molecules, macromolecules such as proteins, or cell surfaces, etc., and are widely used in diagnosis and molecular recognition. , targeted therapy, gene transcription and drug release and other fields. There is a class of aptamers called structure-switching aptamers, which undergo significant conformational changes when interacting with their targets. In recent years, many detection methods based on structure-switching aptamers have emerged. Common detection methods include fluorescence detection, color detect...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/56
Inventor 娄新徽郑冬梅邹如杏
Owner CAPITAL NORMAL UNIVERSITY
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