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A fluorescent biosensor for detecting adenosine triphosphate and its preparation method

A biosensor, adenosine triphosphate technology, used in biochemical equipment and methods, biological testing, microbial determination/inspection, etc., can solve the problems of long detection period, low specificity and sensitivity, and achieve short detection period, easy operation, The effect of reducing complexity

Active Publication Date: 2021-01-22
UNIV OF JINAN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] In order to solve the problems of relatively low specificity and sensitivity and long detection period of the method for detecting ATP in the above prior art, the present invention provides a rolling circle amplification technology and endonuclease with high specificity and sensitivity and fast detection speed. Enzyme feedback amplification fluorescence method to detect ATP biosensor, also relates to its preparation method

Method used

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  • A fluorescent biosensor for detecting adenosine triphosphate and its preparation method
  • A fluorescent biosensor for detecting adenosine triphosphate and its preparation method
  • A fluorescent biosensor for detecting adenosine triphosphate and its preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1 Preparation of Circular Template and Composite Probe

[0057] Prepared with 50 mM Tris-HCl, 10 mM MgCl 2 , T4 DNA Ligase Reaction Buffer with 10 mM DTT and 1 mM ATP. Formulated with 10 mM Na 2 HPO 4 , 10 mM NaH 2 PO 4 , 140 mM NaCl, 1 mM KCl, 1 mM MgCl 2 ,1 mM CaCl 2 , PBS buffer solution of pH=7.4.

[0058] (1) Mix 42 μL sterile water, 6 μL linear template (10 μM), 6 μL ligation probe (10 μM) and 6 μL 10× T4 DNA ligase buffer, denature at 95°C for 5 min, then Slowly cool down to room temperature to complete the hybridization, then add 3 μL of T4 DNA ligase (60 U / μL) to the reaction system, and react at 16°C for 20 hours; after that, the reaction system is placed in a water bath at 65°C for 15 minutes , inactivate the T4 DNA ligase in the system.

[0059] (2) Add 3 μL of exonuclease Ⅰ (20 U / μL) and 3 μL of exonuclease Ⅲ (100 U / μL) to the above reaction system and react at 37°C for 2 h; then put the reaction system at 85°C Heated in a water bath for 1...

Embodiment 2

[0062] A method for preparing a fluorescent biosensor of the present invention, comprising the following steps:

[0063] (1) Mix 2 μL composite probe I (1 μM), 2 μL dNTP (1 mM), 2 μL composite probe II (1 μM), 2 μL Apt-2 (1 μM), 2 μL phi29 DNA polymerase ( 1 U / μL), 2 μL endonuclease IV (0.1 U / μL, 0.2 U / μL, 0.25 U / μL, 0.5 U / μL, 0.75 U / μL, 1 U / μL), 2 μL NMM (2.4 μM) in 2 μL buffer (50 mM Tris-HCl, 10 mM MgCl 2 , 10 mM (NH 4 ) 2 SO 4 , 4 mM DTT, pH 7.5) after mixing, add ATP (100 nM) respectively, mix and react at a constant temperature of 37°C for 90 min;

[0064] (3) Dilute the solution obtained in step (2) with water to 100 μL, and then perform fluorescence detection; the excitation wavelength is set to 486 nm, the emission wavelength is 610 nm, and the detection range is 560 nm-640 nm, and the fluorescence signal changes are read.

[0065] See the test results figure 2 , it can be seen from the figure that as the amount of endonuclease IV increases, the fluorescence in...

Embodiment 3

[0069] A method for preparing a fluorescent biosensor of the present invention, comprising the following steps:

[0070] (1) Mix 2 μL composite probe I (1 μM), 2 μL dNTP (1 mM), 2 μL composite probe II (1 μM), 2 μL Apt-2 (1 μM), 2 μL phi29 DNA polymerase ( Concentrations are 0.1 U / μL, 0.2 U / μL, 0.25 U / μL, 0.5U / μL, 1 U / μL, 1.5 U / μL, 2 U / μL), 2 μL endonuclease IV (0.5 U / μL μL), 2 μL NMM (2.4 μM) in 2 μL buffer (50 mM Tris-HCl, 10 mM MgCl 2 , 10 mM (NH 4 ) 2 SO 4 , 4 mM DTT, pH 7.5) after mixing, add ATP (100 nM) respectively, mix and react at a constant temperature of 37°C for 90 min;

[0071] (3) Dilute the solution obtained in step (2) with water to 100 μL, and then perform fluorescence detection; the excitation wavelength is set to 486 nm, the emission wavelength is 610 nm, and the detection range is 560 nm-640 nm, and the fluorescence signal changes are read.

[0072] See the test results image 3 , it can be seen from the figure that as the amount of phi29 DNA polymer...

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Abstract

The invention relates to the technical field of biosensors, in particular to a fluorescent biosensor for detecting triphosadenine based on rolling circle amplification and incision enzyme feedback amplification. The biosensor comprises two aptamer DNA sequences, a linear padlock probe, a connecting probe, an AP probe, a T4 DNA ligase buffer solution, exonuclease I, exonuclease III, a PBS buffer solution, dNTP, phi29 DNA polymerase and endonuclease IV. A preparation method of the fluorescent biosensor comprises the following steps: (1) constructing an annular template to prepare a compound probe; and (2) combining a compound probe with an incision enzyme and a target object to achieve signal amplification. The probe can be used for achieving detection of high specificity and ultra-sensitivity. The fluorescent biosensor is mild in reaction, fast to detect and good in repeatability.

Description

technical field [0001] The invention relates to the technical field of biosensors, in particular to a fluorescent biosensor for detecting adenosine triphosphate based on a rolling circle amplification technique and an endonuclease feedback amplification method, and also relates to a preparation method thereof. Background technique [0002] Adenosine triphosphate (ATP) is an unstable high-energy compound. ATP is an important energy substance in organisms, which can provide energy for various life activities. The way ATP provides energy is the intramolecular hydrolysis of high-energy phosphate bonds: when the high-energy phosphate bonds in a molecule are hydrolyzed, the energy released is more than twice that of normal chemical bonds, up to 30.54 kJ / mol. Therefore, ATP can store and transfer chemical energy in large quantities in cells. In various biochemical reactions and metabolism of cells, ATP plays an important role in providing energy. Therefore, indicators of organis...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6844G01N33/53
CPCG01N21/6402G01N21/6486
Inventor 黄加栋王敬锋王玉刘素王海旺宋晓蕾张雪赵一菡瞿晓南张儒峰
Owner UNIV OF JINAN
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