An ATP Analysis Method Based on Ratiometric Inorganic Mass Spectrometry Detection

An analysis method and molecular technology, applied in the field of biological and stable isotope sensing, can solve the problems of absolute signal strength fluctuation, absolute strength dependent signal interference, inaccurate detection results, etc., and achieve the effect of simple separation

Active Publication Date: 2022-04-19
SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the presence of various analyte-independent interfering substances, the absolute intensity-dependent signal of conventional bioanalytical methods may be disturbed, resulting in fluctuations in absolute signal intensity and inaccurate detection results

Method used

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  • An ATP Analysis Method Based on Ratiometric Inorganic Mass Spectrometry Detection
  • An ATP Analysis Method Based on Ratiometric Inorganic Mass Spectrometry Detection
  • An ATP Analysis Method Based on Ratiometric Inorganic Mass Spectrometry Detection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] S1: Synthesis of gold nanoparticles by the commonly used sodium citrate reduction method;

[0052] S2: Synthesis of DNA1-magnetic bead complexes and DNA2-AuNPs complexes respectively;

[0053] S3: The two complexes were added to the reaction system in different ratios to form a core-satellite structure, and then 7 μL of 10 mM PBS (PH = 7.4, 150 mM NaCl) and 33 μL of 20 mM Tris-HCl (PH = 7.4, 100 mM NaCl) were added ) Mix and shake for a few minutes.

[0054] S4: Add 20 μL of ATP at a certain concentration to the system, and shake for 4 hours;

[0055]S5: Perform magnetic separation on the reacted solution to obtain a supernatant, which is reacted with 50 μL aqua regia for 20 minutes, and then dissolved by 1.5 ng ml -1 Dilute the standard In solution to 4mL.

[0056] S6: Recording by ICPMS of the 197 Au and 115 In signal intensity and calculate its ratio.

[0057] Experimental results:

[0058] This experiment is to explore the effect of the ratio of the two compl...

Embodiment 2

[0063] S1: Synthesis of gold nanoparticles by the commonly used sodium citrate reduction method;

[0064] S2: Synthesis of DNA1-magnetic bead complexes and DNA2-AuNPs complexes respectively;

[0065] S3: 3 μL DNA1-magnetic bead complex and 6 μL DNA2-AuNPs complex were added to the reaction system to form a core-satellite structure, and then 7 μL 10 mM PBS (PH = 7.4, 150 mM NaCl) and 33 μL 20 mM Tris-HCl (PH = 7.4, 100mM NaCl) Mix and shake for a few minutes.

[0066] S4: Add ATP or other interfering substances to the system, shake and react for 4 hours;

[0067] S5: Perform magnetic separation on the reacted solution to obtain a supernatant, which is reacted with 50 μL aqua regia for 20 minutes, and then dissolved by 1.5 ng ml -1 Dilute the standard In solution to 4mL.

[0068] S6: Recording by ICPMS of the 197 Au and 115 In signal intensity and calculate its ratio.

[0069] Experimental results:

[0070] This experiment is a study on the ATP selectivity of the analytic...

Embodiment 3

[0075] S1: Synthesis of gold nanoparticles by the commonly used sodium citrate reduction method;

[0076] S2: Synthesis of DNA1-magnetic bead complexes and DNA2-AuNPs complexes respectively;

[0077] S3: 3 μL DNA1-magnetic bead complex and 6 μL DNA2-AuNPs complex were added to the reaction system to form a core-satellite structure, and then 7 μL 10 mM PBS (PH = 7.4, 150 mM NaCl) and 33 μL 20 mM Tris-HCl (PH = 7.4, 100mM NaCl) Mix and shake for a few minutes.

[0078] S4: Add different amounts of ATP to the system, shake and react for 4 hours;

[0079] S5: Perform magnetic separation on the reacted solution to obtain a supernatant, which is reacted with 50 μL aqua regia for 20 minutes, and then dissolved by 1.5 ng ml -1 Dilute the standard In solution to 4mL.

[0080] S6: Recording by ICPMS of the 197 Au and 115 In signal intensity and calculate its ratio.

[0081] Experimental results:

[0082] This experiment is to study the analytical performance of the present invent...

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Abstract

The invention discloses an ATP analysis method based on ratio inorganic mass spectrometry (ICPMS) detection. First, the ATP aptamer DNA1 is chemically combined with the magnetic beads through an amide reaction, and a small piece of DNA2 complementary to DNA2 is combined with the gold nanoparticles through the formation of AU‑S bonds. Second, the two complexes were added to the reaction system in a specific ratio to form a core‑satellite structure. When ATP was added to the system, the specific binding of the aptamer to ATP resulted in the release of satellite signals (AuNPs). As the amount of ATP increased, the AuNPs released in the supernatant increased, as detected by ICPMS of the supernatant 197 Au / 115 The In signal ratio becomes higher and higher. because 197 Au / 115 The In signal ratio is related to the corresponding ATP amount, and the present invention passes 197 Au / 115 The signal ratio of In enables the determination of ATP content.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to the field of stable isotope (Metal stable isotope) sensing. Background technique [0002] Adenosine triphosphate (ATP) is the main carrier of chemical energy in living organisms and thus has a key role in the integration and regulation of cellular metabolism. As an indicator of cell damage and cell vitality, ATP-related human diseases include Parkinson's disease, cardiovascular disease, and Alzheimer's disease. Therefore, the detection of ATP is of great significance in clinical diagnosis and biological research. In recent years, a variety of ATP analysis strategies have been proposed through different analysis principles, including colorimetry, fluorescence, chemiluminescence, electrochemical and fluorescence imaging. Despite the great success of scientists, constructing an ATP sensor that can provide accurate and reproducible quantitative results in complex sample matrices remain...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N27/626G01N33/543G01N33/553
CPCG01N27/62G01N33/54326G01N33/54333G01N33/553
Inventor 刘睿王超群吕弋
Owner SICHUAN UNIV
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