CN105548261A - Telomere length detecting method based on biological nano channel of aerolysin - Google Patents
Telomere length detecting method based on biological nano channel of aerolysin Download PDFInfo
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- CN105548261A CN105548261A CN201510877604.9A CN201510877604A CN105548261A CN 105548261 A CN105548261 A CN 105548261A CN 201510877604 A CN201510877604 A CN 201510877604A CN 105548261 A CN105548261 A CN 105548261A
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- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
Abstract
The invention discloses a telomere length detecting method based on a biological nano channel of aerolysin. The method comprises the following steps: (1) opening the G-quadruplet structure of telomere DNA of human; (2) preparing a biological nano channel of aerolysin: (a) activating aerolysin; (b) preparing a phospholipid n-decane solution; (c) preparing phospholipid bilayer; (d) forming a biological nano channel of aerolysin; (3) using the biological nano channel of aerolysin to detect the telomere length: (a) collecting monomolecular signal data; (2) analyzing the monomolecular signal data. Compared with the prior art, the provided method has the advantages that the operation is simple and efficient; enzyme digestion, molecular hybridization, or fluorescence labeling is not needed; the consumption of DNA substrate is little; the detection sensitivity and resolution are high; the price is cheap; the absolute length of telomere can be precisely and directly measured in real time; and the method has a positive meaning for bioscience research.
Description
Technical field
The present invention relates to the DNA single molecular detection technology of life science, particularly a kind of method based on aerolysin biological nano Air conduct measurement telomere length.
Background technology
Telomere is a bit of DNA-protein complex being positioned at end of chromosome, has the effect of stable end of chromosome structure and control CDC.Telomeric dna is then multiple multiple non-transcribed sequences 5 '-(TTAGGG) of one section of strand
n-3 '.Cell often divides once, and the chromosomal telomere of every bar just can lack one section, and when lacking to a certain extent, cell will activate Apoptosis mechanism, namely moves towards apoptosis.Research finds: the length of telomere and the life-span of people and disease have close ties, and therefore, the length measuring telomere is significant to studying life science.
Biological nano passage be modern rise detection nucleic acid, polypeptide, carbohydrate a kind of Single Molecule Detection means, the more albumen hole of application is staphylococcus alpha hemolysin (α-hemolysin), mycobacterium smegmatis (MspA) at present.But due to the restriction in himself aperture, these two kinds of protein channel go back Shortcomings for the small resolution realizing DNA molecular.And aerolysin biological nano passage has less aperture, higher sensitivity, and its inner chamber is positively charged, the via hole speed of the DNA that can effectively slow down, thus high flux, the detection of unmarked, high-sensitive single molecule analysis can be realized.
At present, the method for measuring junction grain length mainly comprises southern blotting technique method, Hybridization Protection Assay method, fluorescence in situ hybridization, quantitative PCR method etc.Described southern blotting technique method needs to use restriction endonuclease DNA digestion, and need to use isotope or biotin labeled telomere specific probe to hybridize with it, quantitatively detect eventually through opacimeter, but the data of the telomere length obtained also comprise the sub-telomere district of part; Therefore, the actual length of telomere cannot be measured by southern blotting technique method.Described Hybridization Protection Assay method needs to use fluorescently-labeled hybridization technique, by detecting its luminous intensity to realize detection; But, adopt Hybridization Protection Assay method cannot obtain the information of telomere profound level, the physical length of telomere can not be measured.Described fluorescence in situ hybridization is not only harsh to sample requirement, and operating process is complicated, needs to carry out marking, hybridizing, and cannot obtain the absolute growth of telomere.Described quantitative PCR method is the ratio repeating the gene of copy ratio and single copy by measuring telomere, and namely T/S value obtains the average length of telomere.But in practical operation, said method all directly cannot measure the length of telomere, both cannot detect the physical length of telomere, needs again to carry out the operations such as enzyme is cut, molecular hyridization, fluorescence labeling, testing process relative complex.
Summary of the invention
The object of the invention is to solve the problem, a kind of method based on aerolysin biological nano Air conduct measurement telomere length is provided, it is cut without the need to enzyme, molecular hyridization and fluorescence labeling, less to the consumption of DNA substrate, detect more real-time high-efficiency, the absolute growth of energy Measurement accuracy telomere.
For achieving the above object, present invention employs following technical scheme.
Based on a method for aerolysin biological nano Air conduct measurement telomere length, it is characterized in that, comprise the following steps:
(1) the G-tetrad structure of mankind's telomeric dna is opened
Mankind's telomeric dna is added 1mL electrolyte solution, and the G-tetrad structure of mankind's telomeric dna is opened under described electrolyte solution condition;
(2) aerolysin biological nano passage is prepared
1. aerolysin is activated
Aerolysin is dissolved in trypsin-EDTA solutions, and the ratio of aerolysin and trypsin-EDTA solutions is 1:100; In constant temperature oven, place 30min at 25 DEG C of temperature, prepare with Tris-HCl damping fluid, pH=8.0 and store in-20 DEG C of refrigerators after activation, storing concentration is 0.1mg/ml;
2. phosphatide n-decane solution is prepared
Be dissolved in n-decane solution by 1,2-bis-phytane acyl phospholipids at normal temperatures, concentration is 25mg/ml;
3. phospholipid bilayer is prepared
Adopt polyacetal resin detection cell, described detection cell contains detection cell A(cis to be held) and detection cell B(trans hold), described detection cell A etches the micropore that diameter is 50 μm (lying prone for phospholipid bilayer attached), then the phosphatide n-decane solution prepared with thin marten hair pen both sides uniform application inside and outside described micropore drying up with nitrogen;
Described detection cell A and described detection cell B is assembled, hold at detection cell A(cis) add the electrolyte solution of 1mL containing mankind's telomeric dna, hold at detection cell B(trans) add 1mL not containing the electrolyte solution of mankind's telomeric dna, a pair Ag/AgCl electrode is immersed in the buffer solution of described detection cell respectively, specify detection cell A(cis end) be zero potential point current potential, apply the transmembrane voltage of 400 ~ 500mv, if immobilized artificial membrane is broken up between this voltage, then think that it is the phospholipid bilayer tunic that can form nanochannel;
4. aerolysin biological nano passage is formed
After preparing phospholipid bilayer, add 3 μ L aerolysin solution nearby at the micropore of described detection cell A, apply the transmembrane voltage of 100mv, aerolysin is held from detection cell A(cis) spontaneous embedding phospholipid bilayer tunic; Form single stable aerolysin biological nano passage, the step that 50 ± 5pA occurs ion current under the concentration and pH value of described electrolyte solution rises;
(3) with aerolysin biological nano Air conduct measurement telomere length
1. single molecule signals data are gathered
The transmembrane voltage of 140mv is applied at the two ends of aerolysin biological nano passage, mankind's telomeric dna can hold from detection cell A(cis under the driving of electric field) worn to detection cell B(trans by aerolysin biological nano passage and hold), and then produce a large amount of with its structure, the corresponding current blocking signal of chain length;
2. single molecule signals data are resolved
To the Xining of the single molecule signals collected, block electric current and resolve:
DNA chain is longer, and the time through aerolysin biological nano passage can be longer, and namely Xining is longer;
Carry out assistant analysis to the current blocking degree of the single molecule signals collected, because DNA molecular not passes described biological nano passage in complete linear pattern, thus DNA chain is longer, and the blocking-up caused nanochannel can be larger, and namely current blocking degree is larger;
The different mankind's telomeric dna of each repetitive sequence number blocks degree value by obtaining with the specific Xining value corresponding to it and specific currents, but when repetitive sequence number >=3, the change that current blocking degree value produces is very faint, should mainly analyze based on Xining value; By analyzing the parameter of the above Xining value and current blocking degree value, the absolute growth of energy Accurate Determining mankind telomeric dna.
Optionally, the electrolyte solution described in step (1) is 1M magnesium chloride (MgCl
2) with 10mM tri-(methylol) aminomethane (Tris), pH=7.4, utilizing constant temperature blender with magnetic force to stir at 25 DEG C of temperature, 20min mixes.In described magnesium chloride solution, because magnesium ion is unfavorable for the formation of G-tetrad structure, the G-tetrad structure of mankind's telomeric dna can be opened under described electrolyte solution condition.
Further, the single molecule signals Data Analysis that described step (3) is 2. described comprises the following steps:
(1) mankind's telomeric dna---5 '-(TTAGGG) of a series of different repetitive sequence number is measured
n-3 ', wherein, n is positive integer; Add up the Xining value corresponding to mankind's telomeric dna of each repetitive sequence number and block current value;
(2) hold at detection cell A(cis) add a certain amount of cell Genome DNA extraction liquid in electrolyte solution, manufacture the real system of the human inner cell that telomere exists, one section of mankind's telomeric dna to be measured is added detection cell A(cis hold), add up all signal datas measured by described system, comprise Xining value and block current value;
(3) Xining value step (2) produced and the Xining value corresponding to mankind's telomeric dna that blocks each repetitive sequence number that current value and step (1) record with block current value and contrast, the repetitive sequence number of surveyed mankind's telomeric dna can be drawn, i.e. the absolute growth of telomere.The above shows that aerolysin biological nano passage technology can not only detect under the buffer solution condition of preparation, also can realize the mensuration of telomere absolute growth in the real system that telomere exists.
The good effect that the present invention is based on the method for aerolysin biological nano Air conduct measurement telomere length is:
Provide a kind of method based on aerolysin biological nano Air conduct measurement telomere length; its good effect is compared with the conventional method: efficiently simple to operate; cut without the need to enzyme; without the need to molecular hyridization and fluorescence labeling; DNA base consumption amount is little, and sensitivity, the resolution of detection are high, cheap; accurate, the real-time absolute growth directly measuring telomere of energy, has very positive meaning to research life science.
Accompanying drawing explanation
Fig. 1 is the monomolecular schematic diagram of aerolysin biological nano Air conduct measurement.
Fig. 2 is the signal graph that embodiment 1 mankind telomeric dna (telomere 1) perforation blocks.
Fig. 3 is the signal graph that embodiment 2 mankind telomeric dna (telomere 2) perforation blocks.
Fig. 4 is the signal graph that embodiment 3 mankind telomeric dna (telomere 3) perforation blocks.
Fig. 5 is the signal graph that embodiment 4 mankind telomeric dna (telomere 4) perforation blocks.
Embodiment
The present invention is based on the embodiment of the method for aerolysin biological nano Air conduct measurement telomere length below in conjunction with accompanying drawing introduction, 5 embodiments are provided.But it is noted that enforcement of the present invention is not limited to following embodiment.
embodiment 1
Based on a method for aerolysin biological nano Air conduct measurement telomere length, comprise the following steps:
(1) the G-tetrad structure of telomeric dna is opened
The mankind's telomeric dna measured is telomere 1---5 '-TTAGGG-3 '.
Telomere 1 is added 1mL electrolyte solution, and described electrolyte solution is 1M magnesium chloride (MgCl
2) with 10mM tri-(methylol) aminomethane buffer solution (Tris), pH=7.4, utilizing constant temperature blender with magnetic force to stir at 25 DEG C of temperature, 20min mixes.
Because the formation of G-tetrad structure is cation selective, different configurations can be formed: in the most frequently used potassium chloride (KCl) solution in different types of alkali metal salt soln, potassium ion is formed with facilitation to G-tetrad structure, and this structure is unfavorable for the accurate detection of nanochannel to DNA, also obstruction can be produced to DNA by nanochannel.
The electrolyte solution adopted due to step (1) is unfavorable for the formation of G-tetrad structure, and the G-tetrad structure of mankind's telomeric dna can be opened under described electrolyte solution condition.
(2) prepare aerolysin biological nano passage, comprise following concrete steps:
1. aerolysin is activated
Aerolysin is dissolved in trypsin-EDTA solutions, and the ratio of aerolysin and trypsin-EDTA solutions is 1:100; In constant temperature oven, place 30min at 25 DEG C of temperature, prepare with Tris-HCl damping fluid (pH=8.0) after activation and store in-20 DEG C of refrigerators, the concentration of storage is 0.1mg/ml.
2. phosphatide n-decane solution is prepared
Be dissolved in n-decane solution by 1,2-bis-phytane acyl phospholipids at normal temperatures, concentration is 25mg/ml.
3. phospholipid bilayer is prepared
Detection cell material adopts polyacetal resin; Described detection cell comprises detection cell A(cis and holds) and detection cell B(trans hold), described detection cell A is etched with diameter be 50 μm, attached micropore of lying prone for phospholipid bilayer, then to dry up with nitrogen with the thin marten hair pen phosphatide n-decane solution that both sides uniform application prepares inside and outside the micropore of detection cell A.
Described detection cell A and described detection cell B is assembled, holds at detection cell A(cis) add the electrolyte solution of 1mL containing described telomere 1, hold at detection cell B(trans) add 1mL not containing the electrolyte solution of mankind's telomeric dna; Again a pair Ag/AgCl electrode is immersed in the buffer solution of described detection cell respectively, specify detection cell A(cis end) be zero potential point current potential, apply the transmembrane voltage of 400 ~ 500mv, if immobilized artificial membrane is broken up between this voltage, then can think that it is the phospholipid bilayer tunic that can form nanochannel.
4. aerolysin biological nano passage is formed
After preparing phospholipid bilayer, add 3 μ L aerolysin solution nearby at the micropore of described detection cell A, apply the transmembrane voltage of 100mv, make it hold from detection cell A(cis) spontaneous embedding phospholipid bilayer tunic; Form single stable aerolysin biological nano passage, the step that 50 ± 5pA occurs ion current under the concentration and pH value of described electrolyte solution rises.
(3) with aerolysin biological nano Air conduct measurement telomere length
1. single molecule signals data are gathered
The transmembrane voltage of 140mv is applied at the two ends of aerolysin biological nano passage, mankind's telomeric dna can hold from detection cell A(cis under the driving of electric field) worn to detection cell B(trans by aerolysin biological nano passage and hold), and then produce a large amount of with its structure, the corresponding current blocking signal of chain length.
2. single molecule signals data are resolved
To the Xining of the single molecule signals collected, block electric current and resolve:
DNA chain is longer, and the time through aerolysin biological nano passage can be longer, and namely Xining is longer.
Carry out assistant analysis to the blocking-up degree of the single molecule signals collected, because DNA molecular not passes described biological nano passage in complete linear pattern, thus DNA chain is longer, and the blocking-up caused nanochannel can be larger, and namely current blocking degree is larger.
The different mankind's telomeric dna of each repetitive sequence number will obtain the specific Xining value corresponding with it and current blocking degree value.
Embodiment 1 is recorded by detection data analysis, and the Xining of telomere 1 is 1.0ms, and blocking-up degree is that 80%(is see Fig. 2).
embodiment 2
Based on a method for aerolysin biological nano Air conduct measurement telomere length, comprise the following steps:
(1) the G-tetrad structure of telomeric dna is opened
Substance is with embodiment 1.Have unlike: the mankind's telomeric dna measured is telomere 2---5 '-(TTAGGG)
2-3.
(2) aerolysin biological nano passage is prepared
Substance is with embodiment 1.Have unlike: hold at described detection cell A(cis) what add is electrolyte solution containing described telomere 2.
(3) with aerolysin biological nano Air conduct measurement telomere length
Substance is with embodiment 1.Have and recorded by detection data analysis unlike: embodiment 2, the Xining of telomere 2 is 3.0ms, and blocking-up degree is that 92%(is see Fig. 3).
embodiment 3
Based on a method for aerolysin biological nano Air conduct measurement telomere length, comprise the following steps:
(1) the G-tetrad structure of telomeric dna is opened
Substance is with embodiment 1.Have unlike: the mankind's telomeric dna measured is telomere 3---5 '-(TTAGGG)
3-3.
(2) aerolysin biological nano passage is prepared
Substance is with embodiment 1.Have unlike: hold at described detection cell A(cis) what add is electrolyte solution containing described telomere 3.
(3) with aerolysin biological nano Air conduct measurement telomere length
Substance is with embodiment 1.Have and recorded by detection data analysis unlike: embodiment 3, the Xining of telomere 3 is 6.0ms, and blocking-up degree is that 95%(is see Fig. 4).
embodiment 4
Based on a method for aerolysin biological nano Air conduct measurement telomere length, comprise the following steps:
(1) the G-tetrad structure of telomeric dna is opened
Substance is with embodiment 1.Have unlike: the mankind's telomeric dna measured is telomere 4---5 '-(TTAGGG)
4-3.
(2) aerolysin biological nano passage is prepared
Substance is with embodiment 1.Have unlike: hold at described detection cell A(cis) what add is electrolyte solution containing described telomere 4.
(3) with aerolysin biological nano Air conduct measurement telomere length
Substance is with embodiment 1.Have and recorded by detection data analysis unlike: embodiment 4, the Xining of telomere 4 is 9.0ms, and blocking-up degree is that 96%(is see Fig. 5).
embodiment 5
Based on a method for aerolysin biological nano Air conduct measurement telomere length, comprise the following steps:
(1) the G-tetrad structure (with embodiment 1) of telomeric dna is opened.
(2) aerolysin biological nano passage (with embodiment 1) is prepared.
(3) with aerolysin biological nano Air conduct measurement telomere length
1. single molecule signals data (with embodiment 1) is gathered
2. resolve single molecule signals data, comprise the following steps:
I, mankind's telomeric dna of a series of different repetitive sequence number is measured---5 '-(TTAGGG)
n-3 ', wherein, n is positive integer; Add up the Xining value corresponding to mankind's telomeric dna of each repetitive sequence number and block current value.
II, hold at detection cell A(cis) add a certain amount of cell Genome DNA extraction liquid in electrolyte solution, manufacture the real system of the human inner cell that telomere exists.
One section of mankind's telomeric dna to be measured is added detection cell A(cis hold), add up all signal datas measured by described system, comprise Xining value and block current value.
III, Xining value step II produced and the Xining value corresponding to mankind's telomeric dna blocking each repetitive sequence number that current value and step I record and block current value and contrast, the repetitive sequence number of surveyed mankind's telomeric dna can be drawn, i.e. the absolute growth of telomere.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the prerequisite not departing from the inventive method; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (3)
1., based on the method for aerolysin biological nano Air conduct measurement telomere length, it is characterized in that, comprise the following steps:
(1) the G-tetrad structure of mankind's telomeric dna is opened
Mankind's telomeric dna is added 1mL electrolyte solution, and the G-tetrad structure of mankind's telomeric dna is opened under described electrolyte solution condition;
(2) aerolysin biological nano passage is prepared
1. aerolysin is activated
Aerolysin is dissolved in trypsin-EDTA solutions, and the ratio of aerolysin and trypsin-EDTA solutions is 1:100; In constant temperature oven, place 30min at 25 DEG C of temperature, prepare with Tris-HCl damping fluid, pH=8.0 and store in-20 DEG C of refrigerators after activation, storing concentration is 0.1mg/ml;
2. phosphatide n-decane solution is prepared
Be dissolved in n-decane solution by 1,2-bis-phytane acyl phospholipids at normal temperatures, concentration is 25mg/ml;
3. phospholipid bilayer is prepared
Adopt polyacetal resin detection cell, described detection cell contains detection cell A(cis to be held) and detection cell B(trans hold), described detection cell A etches the micropore that diameter is 50 μm (lying prone for phospholipid bilayer attached), then the phosphatide n-decane solution prepared with thin marten hair pen both sides uniform application inside and outside described micropore drying up with nitrogen;
Described detection cell A and described detection cell B is assembled, hold at detection cell A(cis) add the electrolyte solution of 1mL containing mankind's telomeric dna, hold at detection cell B(trans) add 1mL not containing the electrolyte solution of mankind's telomeric dna, a pair Ag/AgCl electrode is immersed in the buffer solution of described detection cell respectively, specify detection cell A(cis end) be zero potential point current potential, apply the transmembrane voltage of 400 ~ 500mv, if immobilized artificial membrane is broken up between this voltage, then think that it is the phospholipid bilayer tunic that can form nanochannel;
4. aerolysin biological nano passage is formed
After preparing phospholipid bilayer, add 3 μ L aerolysin solution nearby at the micropore of described detection cell A, apply the transmembrane voltage of 100mv, aerolysin is held from detection cell A(cis) spontaneous embedding phospholipid bilayer tunic; Form single stable aerolysin biological nano passage, the step that 50 ± 5pA occurs ion current under the concentration and pH value of described electrolyte solution rises;
(3) with aerolysin biological nano Air conduct measurement telomere length
1. single molecule signals data are gathered
The transmembrane voltage of 140mv is applied at the two ends of aerolysin biological nano passage, mankind's telomeric dna can hold from detection cell A(cis under the driving of electric field) worn to detection cell B(trans by aerolysin biological nano passage and hold), and then produce a large amount of with its structure, the corresponding current blocking signal of chain length;
2. single molecule signals data are resolved
To the Xining of the single molecule signals collected, block electric current and resolve:
DNA chain is longer, and the time through aerolysin biological nano passage can be longer, and namely Xining is longer;
Carry out assistant analysis to the current blocking degree of the single molecule signals collected, because DNA molecular not passes described biological nano passage in complete linear pattern, thus DNA chain is longer, and the blocking-up caused nanochannel can be larger, and namely current blocking degree is larger;
The different mankind's telomeric dna of each repetitive sequence number blocks degree value by obtaining with the specific Xining value corresponding to it and specific currents, but when repetitive sequence number >=3, the change that current blocking degree value produces is very faint, should mainly analyze based on Xining value; By analyzing the parameter of the above Xining value and current blocking degree value, the absolute growth of energy Accurate Determining mankind telomeric dna.
2. the method based on aerolysin biological nano Air conduct measurement telomere length according to claim 1, is characterized in that, the electrolyte solution described in step (1) is 1M magnesium chloride (MgCl
2) with 10mM tri-(methylol) aminomethane (Tris), pH=7.4, utilizing constant temperature blender with magnetic force to stir at 25 DEG C of temperature, 20min mixes.
3. the method based on aerolysin biological nano Air conduct measurement telomere length according to claim 1, is characterized in that, the 2. described single molecule signals Data Analysis of described step (3) comprises the following steps:
(1) mankind's telomeric dna---5 '-(TTAGGG) of a series of different repetitive sequence number is measured
n-3 ', wherein, n is positive integer; Add up the Xining value corresponding to mankind's telomeric dna of each repetitive sequence number and block current value;
(2) hold at detection cell A(cis) add a certain amount of cell Genome DNA extraction liquid in electrolyte solution, manufacture the real system of the human inner cell that telomere exists, one section of mankind's telomeric dna to be measured is added detection cell A(cis hold), add up all signal datas measured by described system, comprise Xining value and block current value;
(3) Xining value step (2) produced and the Xining value corresponding to mankind's telomeric dna that blocks each repetitive sequence number that current value and step (1) record with block current value and contrast, the repetitive sequence number of surveyed mankind's telomeric dna can be drawn, i.e. the absolute growth of telomere.
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| CN113138223A (en) * | 2021-04-23 | 2021-07-20 | 华东理工大学 | Single-molecule detection method for perfluoro and polyfluoroalkyl pollutants |
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| CN108181358A (en) * | 2017-11-15 | 2018-06-19 | 华东理工大学 | Method based on aerolysin nanochannel detection amino acid |
| CN108195899A (en) * | 2017-11-15 | 2018-06-22 | 华东理工大学 | The method that biological sulfhydryl compound is detected based on aerolysin nanochannel |
| CN108195899B (en) * | 2017-11-15 | 2020-01-07 | 华东理工大学 | Method for detecting biological sulfhydryl compound based on aerolysin nanochannel |
| CN108181358B (en) * | 2017-11-15 | 2020-02-11 | 华东理工大学 | Method for detecting amino acid based on aerolysin nanochannel |
| CN109187674A (en) * | 2018-07-17 | 2019-01-11 | 广东工业大学 | A kind of lossless detection method of micron openings through-hole wall quality |
| CN109187674B (en) * | 2018-07-17 | 2020-06-19 | 广东工业大学 | Nondestructive testing method for quality of inner wall of through hole with micron hole |
| CN109557157A (en) * | 2018-12-05 | 2019-04-02 | 中国科学院重庆绿色智能技术研究院 | The nanometer pore single-molecule system and its monitoring method of a kind of dynamic monitoring G- tetrad forming process and application |
| CN112480204A (en) * | 2020-04-13 | 2021-03-12 | 南京大学 | Protein/polypeptide sequencing method adopting Aerolysin nanopores |
| CN113138223A (en) * | 2021-04-23 | 2021-07-20 | 华东理工大学 | Single-molecule detection method for perfluoro and polyfluoroalkyl pollutants |
| CN113138223B (en) * | 2021-04-23 | 2022-10-28 | 华东理工大学 | Single-molecule detection method for perfluoro and polyfluoroalkyl pollutants |
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Application publication date: 20160504 |