Application of Aerolysin Nanopore Channels in Biophosphorylation and Related Enzyme Analysis

An Aeromonas lysin and nanopore technology, applied in the biological field, can solve the problems of weak interaction and insensitive detection of phosphorylation state, and achieve the effects of real-time monitoring, high sensitivity and convenient detection.

Active Publication Date: 2022-03-25
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 Channels in Biophosphorylation and Related Enzyme Analysis
  • Application of Aerolysin Nanopore Channels in Biophosphorylation and Related Enzyme Analysis
  • Application of Aerolysin Nanopore Channels in Biophosphorylation and Related Enzyme Analysis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1: Detection of nucleic acid phosphorylation / dephosphorylation

[0047] (1) Construction of Aeromonas lysin nanopore channel: Assemble the detection cell and add pH=8 electrolyte solution (Tris buffer, 1 mol / L) at both ends of the detection cell, and 50 μm micropores on the detection cell A phospholipid bilayer is constructed at the phospholipid bilayer, and aeromonasin is added to one end of the detection cell, and the aerolysin nanopores are formed on the phospholipid bilayer;

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

[0049] (3) Compare and analyze the blocking current signal...

Embodiment 2

[0053] Example 2: Detection of alkaline phosphatase

[0054] (1) Construction of Aeromonas lysin nanopore channel: Assemble the detection cell and add MgCl containing MgCl to both ends 2 pH=7.5 electrolyte solution (Tris buffer, 1mol / L), MgCl 2 The concentration of 20mmol / L, build a phospholipid bilayer at the 50μm micropore in the detection cell, add Aeromonasin at one end of the detection cell, and wait for it to form Aeromonasin on the phospholipid bilayer. nanopore;

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

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

[0057] Described analyte is re...

Embodiment 3

[0060] Example 3: Detection of Polypeptide Phosphorylation

[0061] (1) Construction of Aeromonas lysin nanopore channel: Assemble the detection cell and add MgCl containing MgCl to both ends 2 pH=7.5 electrolyte solution (Tris buffer, 1mol / L), MgCl 2 The concentration of 20 mmol / L, build a phospholipid bilayer at the 50μm micropore in the detection cell, add Aeromonasin at one end of the detection cell, and wait for it to form Aeromonasin 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 (Cis end) of the detection cell, and under the drive of the potential, the analyte passes through the Aeromonas lysin nanopore to generate a blocking current Signal and blocking current time.

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

[0064] The analyte uses LRRASLG as...

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Abstract

The invention discloses the application of an aerolysin nanopore channel in the analysis of biological phosphorylation and related enzymes. Specifically, the aerolysin nanopore channel is constructed, and voltage is applied to both ends of the constructed aerolysin nanopore channel, and the analyte or analyte-related probe molecules are added to one end of the detection cell, Driven by the voltage, the analyte passes through the aerolysin nanopore to generate a blocking current signal and the blocking current time. By comparing and analyzing the blocking current signal and the blocking current time, the corresponding molecular weight of the analyte is obtained. Detection information. The invention discloses a new application of the aerolysin nanopore channel, which can be used for the phosphorylation detection and analysis of nucleic acid, polypeptide and protein, does not require DNA motor protein, has high sensitivity, is convenient for detection, and can further realize the Activity analysis and quantitative analysis of various enzymes such as kinases, phosphatases, and enzyme inhibitors, and real-time monitoring of enzyme activities can be realized.

Description

technical field [0001] The invention belongs to the field of biotechnology, and particularly relates to the application of Aeromonas lysin nanopores in biological phosphorylation and related enzyme analysis. Background technique [0002] Phosphorylation and dephosphorylation of DNA or protein are two very important processes in nucleic acid metabolism. Among them, abnormal phosphorylation at the 3' end of DNA or dephosphorylation at the 5' end of DNA is associated with diseases such as Alzheimer's disease and cancer. Therefore, accurate analysis of DNA or protein phosphorylation and dephosphorylation is crucial. 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 detection methods for DNA or protein phosphorylation and dephosphorylation mainly include high-resolution mass spectrometry, fluorescence and radioisotope l...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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