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Biosensor for detecting adenosine triphosphate (ATP)

A technology of biosensor and adenosine triphosphate, which is applied in the direction of instruments, measuring devices, scientific instruments, etc., can solve the problems of long detection cycle, low specificity and sensitivity, etc., and achieve the effect of short detection cycle, good specificity and low process cost

Inactive Publication Date: 2018-06-01
UNIV OF JINAN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] 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 dual-purpose method based on the assistance of exonuclease III with high specificity and sensitivity and fast detection speed. Biosensor for detecting ATP by amplified fluorescence method

Method used

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  • Biosensor for detecting adenosine triphosphate (ATP)
  • Biosensor for detecting adenosine triphosphate (ATP)
  • Biosensor for detecting adenosine triphosphate (ATP)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 The effect of different concentrations of Exo III on the detection of ATP fluorescence intensity.

[0036] (1) Add 14 μL sterilized water, 2 μL Exo III enzyme buffer, 2 μL 100 μM Aptamer chain and 2 μL 100 μM Trigger chain into the pre-prepared sterilized EP tube; shake for 30s, at 95°C Incubate for 5 minutes, cool slowly to room temperature, and store at -20°C for use.

[0037] (2) Add 6 μL of the solution in step (1) to the EP tube, followed by adding ATP (5 μL, 10 2 nM), HAP (3 μL, 5 μM), MB (6 μL, 10 μM), 3 μL Exo III (10 U / mL, 25 U / mL, 50 U / mL, 75 U / mL, 100 U / mL, respectively mL, 125 U / mL, 150 U / mL), 6 μL ExoIII enzyme buffer and 31 μL sterile water. Shake for 30 s and incubate for 2 h in a 37°C incubator.

[0038] (3) Dilute 60 μL of the solution after the reaction in the previous step to 70 μL, set the excitation wavelength of the fluorometer to 486 nm, the emission wavelength to 518 nm, and the detection range of 500 nm-650 nm to read the changes i...

Embodiment 2

[0040] Example 2 Effects of different concentrations of HAP on detection of ATP fluorescence intensity.

[0041] (1) Add 14 μL sterilized water, 2 μL Exo III enzyme buffer, 2 μL 100 μM Aptamer chain and 2 μL 100 μM Trigger chain into the pre-prepared sterilized EP tube; shake for 30s, at 95°C Incubate for 5 minutes, slowly cool down to room temperature, and store at -20 °C for use;

[0042] (2) Add 6 μL of the solution in step (1) to the EP tube, followed by adding 3 μL of HAP (final concentrations are 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM), ATP (5 μL10 2 nM), MB (6 μL, 5 μM), exonuclease III (3 μL, 100U / mL), 6 μL ExoIII enzyme buffer and 31 μL sterilized water. Shake for 30 s and incubate in a 37°C incubator for 2 h;

[0043] (3) Dilute 60 μL of the solution after the reaction in the previous step to 70 μL, set the excitation wavelength of the fluorometer to 486nm, the emission wavelength to 518nm, and the detection range to 500 nm-650 nm, and read the change of the fluoresc...

Embodiment 3

[0045]Example 3 The effect of different concentrations of MB on the detection of ATP fluorescence intensity.

[0046] (1) Add 14 μL sterilized water, 2 μL Exo III enzyme buffer, 2 μL 100 μM Aptamer chain and 2 μL 100 μM Trigger chain into the pre-prepared sterilized EP tube; shake for 30s, at 95°C Incubate for 5 minutes, slowly cool down to room temperature, and store at -20 °C for use;

[0047] (2) Add 6 μL of the solution in step (1) into the EP tube, followed by adding 6 μL of MB (final concentrations are 2 μM, 4 μM, 6 μM, 8 μM, 10 μM, 12 μM), ATP (5 μL10 2 nM), HAP (3 μL, 5 μM), Exo III enzyme (3 μL, 100U / mL), 6 μL ExoIII enzyme buffer and 31 μL sterilized water. Shake for 30s, and incubate for 2 hours in a 37°C incubator;

[0048] (3) Dilute 60 μL of the solution after the reaction in the previous step to 70 μL, set the excitation wavelength of the fluorometer to 486 nm, the emission wavelength to 518 nm, and the detection range of 500 nm-650 nm to read the changes in...

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Abstract

The invention provides a biosensor for detecting adenosine triphosphate (ATP). The biosensor comprises exonuclease III, a triggering chain, an ATP exonuclease, a hairpin probe and a molecular beacon,wherein the ATP content in a solution can be detected through fluorescence; the excitation wavelength of the fluorescence is 486nm; the emission wavelength is 518nm; the detection ranges is 500-650nm.The biosensor is high in specificity, high in sensitivity, mild in reaction conditions, and high in repeatability; the detection method is simple and convenient to operate; the detection period is short; the main detection process is performed in homogeneous phase, so that the reaction speed is increased, and the complexity of operation is reduced.

Description

technical field [0001] The invention belongs to the technical field of biosensors, in particular to a fluorescent biosensor for detecting adenosine triphosphate (ATP) based on nucleic acid aptamers. Background technique [0002] Adenosine triphosphate (ATP) exists in all organisms from microorganisms to higher animals and plants, and is the direct source of energy for various life activities, but its content in the body is not high, and the molar concentration of ATP in cells is usually 1 -10 mM. The main function of ATP in cells is to provide energy, participate in the metabolism of fat, protein, sugar and nucleic acid, and play an irreplaceable role in maintaining the normal function of organisms. ATP is an important energy component of living cells. Dead cells cannot detect ATP, and the ATP metabolism of cancer cells is particularly active. By measuring the content of ATP in the cells, not only the state of the cells can be observed, but also the effects of different ex...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64
CPCG01N21/6486
Inventor 刘素冷雪琪王玉黄加栋
Owner UNIV OF JINAN
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