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A method for the detection of biothiol compounds based on aerolysin nanochannels

A technology for aerolysin and sulfhydryl compounds, which is applied in the field of detecting biological sulfhydryl compounds based on aeromonasin nanochannels, can solve problems such as difficulty in simultaneous detection, and achieves low price, active clinical treatment, and convenient operation. Effect

Inactive Publication Date: 2020-01-07
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the method for detecting biothiol compounds mainly uses fluorescent probes for detection; however, most fluorescent probes can only detect one type of biothiol compounds
It is still difficult to realize the simultaneous detection of the above three biothiol compounds

Method used

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  • A method for the detection of biothiol compounds based on aerolysin nanochannels
  • A method for the detection of biothiol compounds based on aerolysin nanochannels

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] A method for detecting biological thiol compounds based on aerolysin nanochannels for detecting cysteine, comprising the following steps:

[0044] (1) Design a DNA probe connected with an alkynyl group; the base length of the DNA probe is 4; the alkynyl group is modified in the middle position of four base nucleotides, which can be combined with the mercapto group of the mercapto compound Carry out addition reaction, finally form DNA probe-sulfhydryl compound adduct:

[0045]

[0046] (2) Mix 20 μL, 200 μM, pH value of 8.0 DNA probe solution with 10 μL, 1 mM, pH value of 8.0 cysteine ​​solution, and irradiate with 320-400 nm ultraviolet light for 1-4 hours. The addition reaction of the alkyne group to the sulfhydryl group on the cysteine ​​links the cysteine ​​to the DNA probe.

[0047] (3) Preparation of aerolysin bionanochannels

[0048] ① Activated aerolysin

[0049]Dissolve aerolysin in trypsin-EDTA solution, place it in an incubator at 25°C for 30 minutes for...

Embodiment 2

[0068] A method for detecting biosulfhydryl compounds based on aerolysin nanochannels is used for detecting homocysteine, and the basic steps are the same as those in Example 1.

[0069] The difference is:

[0070] (1) (with embodiment 1).

[0071] (2) Mix 20 μL, 200 μM, pH value of 8.0 DNA probe solution with 10 μL, 5 mM, pH value of 8.0 homocysteine ​​solution, and irradiate with 365 nm ultraviolet light for 2 hours. The addition reaction of the sulfhydryl group on homocysteine ​​attaches homocysteine ​​to the DNA probe.

[0072] (3) (with embodiment 1).

[0073] (4) Detection of DNA-homocysteine ​​with aerolysin

[0074] Acquisition of DNA probe-homocysteine ​​single-molecule characteristic signal-this signal is a single-molecule signal that can reflect the characteristics of homocysteine.

[0075] Example 2 Obtaining the characteristic blocking current of DNA probe-homocysteine ​​single molecule is I / I 0 =0.30-0.35; where, I 0 is the opening current value; I is the b...

Embodiment 3

[0077] A method for detecting biosulfhydryl compounds based on the aerolysin nanochannel is used for detecting glutathione, and the basic steps are the same as those in Example 1.

[0078] The difference is:

[0079] (1) (with embodiment 1).

[0080] (2) Mix 20 μL, 200 μM, pH 8.0 DNA probe solution with 10 μL, 5 mM, pH 8.0 glutathione solution, and irradiate with 365 nm ultraviolet light for 2 hours to utilize the alkynyl and glutathione groups on the probe to The addition reaction of the sulfhydryl group on the glutathione links the glutathione to the DNA probe.

[0081] (3) (with embodiment 1).

[0082] (4) Detection of DNA probe-glutathione with aerolysin

[0083] Acquisition of DNA probe-glutathione single-molecule signature signal—this signal is a single-molecule signal that can reflect the signature of glutathione.

[0084] Example 3 obtains the characteristic blocking current of DNA probe-glutathione single molecule as I / I 0 =0.20~0.30; Among them, I 0 is the open...

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Abstract

The invention relates to a method for detecting amino acids based on an aerolysin nanometer channel. The method comprises: (1) designing a DNA probe linked to alkynyl; (2) linking a mercapto compoundto the DNA probe; (3) preparing an aerolysin biological nanometer channel: a) activating aerolysin, b) preparing a phospholipid n-decane solution, c) preparing a phospholipid bilayer, and d) forming the aerolysin biological nanometer channel; and (4) detecting the biological mercapto compound by using aerolysin: a) collecting the single-molecule signal of the DNA probe, b) collecting the single-molecule signal of the biological mercapto compound, c) collecting the single-molecule signal of the DNA probe-mercapto compound, and d) carrying out statistics on the collected single-molecule signal of the DNA probe-mercapto compound to obtain the characteristic information of the mercapto compound, and simultaneously detecting different mercapto compounds based on the obtained characteristic information of the mercapto compound. With the method of the present invention, the ultra-sensitive resolution at the single molecule level can be achieved, the operation is convenient, real-time and accurate, and the positive significance is provided for life science research.

Description

technical field [0001] The invention relates to a detection technology for analyzing and detecting single molecules in life chemistry, in particular to a method for detecting biological sulfhydryl compounds based on an aerolysin nanochannel. [0002] technical background [0003] Biological nanochannels are a single-molecule detection method emerging in modern times to detect nucleic acids, polypeptides, and sugars. Currently, the most widely used protein pores are Staphylococcus α-hemolysin (α-hemolsyin) and Mycobacterium smegmatis (MspA). However, due to the limitation of the pore size of the protein pore itself, the above two protein channels are not enough to realize the micro resolution of DNA molecules. The aerolysin biological nanochannel has a smaller pore size, higher sensitivity, and its inner cavity is positively charged, which can effectively slow down the passing rate of DNA, thereby achieving high throughput, label-free, and high sensitivity. , DNA probe-based...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N27/26
CPCG01N27/26
Inventor 应佚伦孟福娜张隽佶李自远龙亿涛
Owner EAST CHINA UNIV OF SCI & TECH
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