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Application of aerolysin nanopore channel in biological phosphorylation and related enzyme analysis

An aerolysin and nanopore technology, applied in the biological field, can solve the problems of insufficient sensitivity of phosphorylation state detection and weak interaction, and achieve real-time monitoring, high sensitivity and convenient detection

Active Publication Date: 2020-11-10
NANJING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, previous reports used α-hemolysin as a nanopore, but the interaction between the nanopore and DNA molecules is weak, and DNA molecules can easily pass through the nanopore and the detection of phosphorylation status is not sensitive enough.

Method used

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  • Application of aerolysin nanopore channel in biological phosphorylation and related enzyme analysis
  • Application of aerolysin nanopore channel in biological phosphorylation and related enzyme analysis
  • Application of aerolysin nanopore channel in biological phosphorylation and related enzyme analysis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1: Nucleic acid phosphorylation / dephosphorylation detection

[0047] (1) Construct the aerolysin nanopore channel: Assemble the detection cell and add an electrolyte solution (Tris buffer, 1mol / L) with pH=8 at both ends of the detection cell, and place a 50 μm micropore in the detection cell Build a phospholipid bilayer at one end of the detection cell, add aerolysin to one end of the detection cell, and wait for it to form aerolysin nanopores on the phospholipid bilayer;

[0048] (2) Apply a voltage of 300mV at both ends of the aerolysin nanopore channel, add 10ul of the analyte (the amount to be added depends on the detection requirements, generally 1-100uL) into one end of the detection cell (Cis end), Driven by the potential, the analyte passes through the aerolysin nanopore to generate a blocking current signal and blocking current time;

[0049] (3) Comparing and analyzing the blocking current signal and the blocking current time to obtain the correspondi...

Embodiment 2

[0053] Embodiment 2: the detection of alkaline phosphatase

[0054] (1) Construct the aerolysin nanopore channel: assemble the detection cell and add MgCl-containing 2 Electrolyte solution (Tris buffer, 1mol / L) of pH=7.5, MgCl 2 The concentration is 20mmol / L, build a phospholipid bilayer at the 50μm micropore in the detection pool, add aerolysin at one end of the detection pool, and wait for it to form aerolysin on the phospholipid bilayer Nanopore;

[0055] (2) Apply a voltage of 100mV at both ends of the Aerolysin nanopore channel, add 10ul of the analyte to one end of the detection cell (Cis end), and the analyte passes through the Aeromonas lysate under the drive of the potential. Prime nanopores generate blocking current signals;

[0056] (3) Comparing and analyzing the blocking current signal and the blocking current time to obtain the corresponding detection information of the object to be tested.

[0057] The analytes are phosphorylated DNA strands (P-5'-dA 14 -3'...

Embodiment 3

[0060] Example 3: Detection of polypeptide phosphorylation

[0061] (1) Construct the aerolysin nanopore channel: assemble the detection cell and add MgCl-containing 2 Electrolyte solution (Tris buffer, 1mol / L) of pH=7.5, MgCl 2 The concentration is 20mmol / L, build a phospholipid bilayer at the 50μm micropore in the detection pool, add aerolysin at one end of the detection pool, and wait for it to form aerolysin on the phospholipid bilayer Nanopore;

[0062](2) Apply a voltage of 300mV at both ends of the nanochannel, add 10ul of the analyte to one end of the detection cell (Cis end), and under the drive of the potential, the analyte passes through the aerolysin nanopore to generate a blocking current Signal and blocking current time.

[0063] (3) Comparing and analyzing the blocking current signal and the blocking current time to obtain the corresponding detection information of the object to be tested.

[0064] The analyte uses LRRASLG as a model polypeptide, respectivel...

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Abstract

The invention discloses an application of an aerolysin nanopore channel in biological phosphorylation and related enzyme analysis. Specifically, the method comprises the steps: constructing an aerolysin nanopore channel, applying a voltage to the two ends of the constructed aerolysin nanopore channel, adding a to-be-detected object or related probe molecules of the to-be-detected object to one endof a detection pool, and driving the to-be-detected object by the voltage to penetrate through aerolysin nanopores to generate a blocking current signal and blocking current time; and performing comparative analysis on the blocking current signal and the blocking current time to obtain corresponding detection information of the molecule to be detected. The invention discloses a new application ofan aerolysin nanopore channel, which can be used for phosphorylation detection and analysis of nucleic acid, polypeptide and protein, does not need DNA motor protein, has high sensitivity, is convenient to detect, can further realize activity analysis and quantitative analysis of various enzymes such as kinase, phosphatase and enzyme inhibitor, and can realize real-time monitoring of enzyme activity.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to the application of aerolysin nanopores in the analysis of biological phosphorylation and related enzymes. Background technique [0002] Phosphorylation and dephosphorylation of DNA or protein are two very important processes in nucleic acid metabolism. Among them, abnormal phosphorylation of DNA 3' end or dephosphorylation of 5' end are related to diseases such as Alzheimer's disease and cancer. Therefore, accurate analysis of DNA or protein phosphorylation and dephosphorylation is very important. Among them, DNA phosphorylation and dephosphorylation mainly include four different states: 5' end phosphorylation, 5' end dephosphorylation, 3' end phosphorylation and 3' end dephosphorylation. At present, the methods for detecting phosphorylation and dephosphorylation of DNA or protein mainly include high-resolution mass spectrometry, fluorescence and radioactive isotope lab...

Claims

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Application Information

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IPC IPC(8): G01N27/48
CPCG01N27/48
Inventor 龙亿涛应佚伦蒋杰李孟寅杨洁于汝佳
Owner NANJING UNIV
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