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Fluorescence sensation method for detecting exonuclease I

An exonuclease and fluorescence sensing technology is applied in the field of fluorescence biosensors to achieve accurate results, less reagent consumption and good selectivity.

Active Publication Date: 2017-09-22
上海柏创翌生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently, no fluorescent sensor for detecting Exo I activity based on the FRET phenomenon of iridium complexes and CCPs has been reported.

Method used

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  • Fluorescence sensation method for detecting exonuclease I
  • Fluorescence sensation method for detecting exonuclease I
  • Fluorescence sensation method for detecting exonuclease I

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] The technical solution adopted by the present invention to solve the above-mentioned technical problems is: a kind of fluorescence sensing method that detects Exo I, concrete steps are as follows:

[0027] (1) Mix 90 μL of 3.28 μM CCP with 1 μL of 1 mM iridium complex, and then add a corresponding volume of 10×Exo I buffer to obtain 100 μL of fluorescence based on the FRET effect of CCP and iridium complexes for detection of Exo I sensor system. The fluorescence intensity was detected, and the ratio of the fluorescence intensity of the CCP (415nm) to the fluorescence intensity of the iridium complex (530nm) was calculated.

[0028] (2) Add 2 μL of 10 μM ssDNA to the system in step (1), detect the change of fluorescence intensity, and calculate the ratio of the fluorescence intensity of CCP (415nm) to the fluorescence intensity of the iridium complex (530nm).

[0029] (3) Different concentrations of Exo I were added to step (2), and then the corresponding volume of 10×E...

Embodiment 2

[0031] The technical solution adopted by the present invention to solve the above-mentioned technical problems is: a kind of fluorescence sensing method that detects Exo I, concrete steps are as follows:

[0032] (1) Mix 85 μL of 4 μM CCP with 2 μL of 1 mM iridium complex, and then add a corresponding volume of 10× Exo I buffer to obtain 100 μL of a fluorescent sensor based on the FRET effect of CCP and iridium complexes for detecting Exo I system. The fluorescence intensity was detected, and the ratio of the fluorescence intensity of the CCP (415nm) to the fluorescence intensity of the iridium complex (530nm) was calculated.

[0033] (2) Add 1 μL of 15 μM ssDNA to the system in step (1), detect the change of fluorescence intensity, and calculate the ratio of the fluorescence intensity of CCP (415nm) to the fluorescence intensity of the iridium complex (530nm).

[0034](3) Different concentrations of Exo I were added to step (2), and then the corresponding volume of 10×Exo I ...

Embodiment 3

[0036] The technical solution adopted by the present invention to solve the above-mentioned technical problems is: a kind of fluorescence sensing method that detects Exo I, concrete steps are as follows:

[0037] (1) Mix 95 μL of 3 μM CCP with 0.5 μL of 2 mM iridium complex, and then add a corresponding volume of 10× Exo I buffer to obtain 100 μL of fluorescence based on the FRET effect of CCP and iridium complexes for detection of Exo I sensor system. The fluorescence intensity was detected, and the ratio of the fluorescence intensity of the CCP (415nm) to the fluorescence intensity of the iridium complex (530nm) was calculated.

[0038] (2) Add 3 μL of 5 μM ssDNA to the system in step (1), detect the change of fluorescence intensity, and calculate the ratio of the fluorescence intensity of CCP (415nm) to the fluorescence intensity of the iridium complex (530nm).

[0039] (3) Different concentrations of Exo I were added to step (2), and then the corresponding volume of 10×Ex...

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Abstract

The invention discloses a fluorescence sensation method for detecting exonuclease I (Exo I). The fluorescence sensation method is characterized by comprising the following specific steps: (1) mixing a cation conjugated polymer (CCP) with an iridium complex, further adding an Exo I buffer solution so as to obtain a fluorescence sensor system for detecting Exo I and based on a fluorescence resonance energy transfer (FRET) effect of the CCP and the iridium complex, and detecting fluorescence intensity; (2) adding single-chain DNA (ssDNA) into the system of the step (1), and detecting variation of the fluorescence intensity; (3) adding Exo I of different concentrations into the step (2), and detecting activity of the Exo I in a sample. The fluorescence sensation method has the advantage that an analysis sensation method which is good in specificity, high in sensitivity, rapid in detection speed and accurate and reliable in result for Exo I detection is developed for a first time.

Description

technical field [0001] The invention relates to a fluorescent biosensor, in particular to a fluorescent sensing method for detecting exonuclease I (Exo I). Background technique [0002] Exonucleases are a class of enzymes that catalyze the degradation of nucleotides from the beginning of a polynucleotide chain. Exonucleases are divided into: exonuclease I (Exo I) and exonuclease III (Exo III). Exo I is an enzyme that can only catalyze the hydrolysis of phosphodiester bonds between bases one by one from the 3' end to the 5' end of single-stranded DNA (ssDNA) according to the base order, and gradually degrade ssDNA. After Exo I hydrolyzes ssDNA, the final product is deoxyribonucleotides one by one. Exo I does not require the base sequence of ssDNA, and does not have the ability to hydrolyze double-stranded DNA. Exonucleases play an important role in many biological processes, such as DNA replication, recombination, and repair. The most important function is to maintain the...

Claims

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Application Information

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IPC IPC(8): G01N21/64
Inventor 胡宇芳张青青杜春暖王娇饶家佳郭智勇葛国平王邃
Owner 上海柏创翌生物技术有限公司
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