CN107436317A - A kind of method using nanochannel technology for detection cancer - Google Patents
A kind of method using nanochannel technology for detection cancer Download PDFInfo
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- CN107436317A CN107436317A CN201710599870.9A CN201710599870A CN107436317A CN 107436317 A CN107436317 A CN 107436317A CN 201710599870 A CN201710599870 A CN 201710599870A CN 107436317 A CN107436317 A CN 107436317A
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
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Abstract
The present invention's provides a kind of method using nanochannel technology for detection cancer, the microRNA mark related by detecting cancer, the early detection of cancer is realized, to fill up the blank of the project research field.Empirical tests, Tumor biomarkers microRNA can simply, quickly be detected using this method, and microRNA species can be judged according to its blocking time, current amplitude etc..
Description
Technical field
The invention belongs to nanochannel gene sequencing technology field, and in particular to one kind utilizes nanochannel technology for detection cancer
The method of disease.
Background technology
Nanochannel detection technique is to insert α hemolysin in lipid bilayer to form natural nanochannel, nanometer
Passage is connected with two fluid pools, when applying voltage to nanochannel two end electrodes, can produce a constant benchmark ion
Electric current.When nucleotide sequence by when, due to physics occupy-place, it will change the resistance of nanochannel, the change of resistance will cause from
The change of electron current, form the modulation electric current of similar square-wave signal.The waveform of modulation electric current and the physical message of biomolecule are straight
Correlation is connect, analyzes amplitude, the duration of square-wave signal, with regard to that can recognize nucleotide sequence length and classification, this can be considered a kind of day
So detect monomolecular sensor（See Fig. 1）.MicroRNA plays carcinogenic or suppression cancer effect, by adjusting machine after transcription
System, organism physiology and pathologic process are participated in, generation, development, transfer and the prognosis to tumour are related, can be as the biology mark of cancer
Will and treatment prognostic indicator.It is considered as the new development for detecting cancer field using nanochannel detection technique detection microRNA
One of direction, once field technology innovation will be carried out to medical science detection band by obtaining substantive breakthroughs.The present invention is led to based on nanometer
Road detection technique, for it is quick, accurate, be realized with a low cost early diagnosis of cancer new method be provided, led with filling up the project research
The blank in domain.
The content of the invention
It is an object of the invention to provide a kind of method using nanochannel technology for detection cancer, by detecting cancer phase
The microRNA marks of pass, realize the early detection of cancer.The present invention provides detailed experimental procedure and optimal hair answers bar
Part, to fill up the blank of the project research field.The technical solution used in the present invention is as follows.
A kind of method using nanochannel technology for detection cancer, this method need to use a kind of sample cell, sample cell point
For two compartments of cis and trans（B, B'）, kept apart by 120-159 μm of Toflon films between compartment.Hole A and hole A'
It is for first time liquid feeding body and insertion electrode, is connected by electrode with patch-clamp, chemical signal is converted into electric signal, it
The addition of liquid operates from lower face C and hole C' afterwards.This method comprises the following steps：
（1）Magneton is respectively put into chamber B and chamber B', then ultra-pure water is injected from hole A and hole A' respectively, with liquid level height
It is defined in micron openings, calculates the volume 2mL of required ultra-pure water；
（2）Magneton is taken out, pours out ultra-pure water, and rinses Toflon films, chamber and hole repeatedly, is finally rushed with absolute ethyl alcohol
Wash；
（3）After rinsing well, the drop remained in chamber and on Toflon films is dried up with nitrogen；
（4）Then, drip about 10 ~ 15 μ L H liquid of drop respectively at micron openings on Toflon films both sides, and blow open rapidly,
Make it on film extend to scatter.Wherein, H liquid is the mixed liquor of 1mL pentanes and 100 μ L hexadecanes；
（5）Be respectively put into magneton in chamber B and chamber B', then, from hole A and hole A' respectively with pipettor add 0.8 ~
1.1mL contains 10 mmol/ L Tris and 1 mol/ L KCl buffer solutions, then inserts Ag/AgCl electrodes respectively；
（6）Then, hole A electrode is connected with the trans ends popped one's head in, and hole A' electrode is connected with the cis ends popped one's head in；
（7）15 ~ 25 μ L L liquid are added in chamber B and chamber B' respectively with 25 μ L micropipettor, make L liquid equal on liquid level
Spread out evenly, then wait 30s-1min.Wherein, L liquid is the rapid featheriness of mixed liquor of 25mg phosphatide and 2.5mL pentanes；
（8）Add 0.8 ~ 1.1mL 10 mmol/ L Tris buffer solutions respectively from hole C and hole C', see whether shape
Into phospholipid bilayer, such as formed, then carry out subsequent experimental；As do not formed, then blown and beaten repeatedly several times, directly from hole C and hole C' ends
Phospholipid bilayer is formed to it；
（9）After phospholipid bilayer is formed, apply the mV of voltage+180 mV ~+200 to system, alpha hemolysis is added in the B' of hole
Its final concentration of 0.0125 ~ 0.145ng/mL of plain solution, and open hole B' stirring system, control suitable rotating speed, stir about
2min, form nanometer single channel；
（10）After forming nanochannel, a certain amount of microRNA is added in the B' of hole, open stirring system, after stir about 5s,
Start to collect detection signal.
Empirical tests, Tumor biomarkers microRNA can simply, quickly be detected using this method, and can basis
Its blocking time, current amplitude etc. judge microRNA species.
Brief description of the drawings
Fig. 1 nanochannel technical principle schematic diagrames.
Fig. 2 sample pool structure schematic diagrames.
Fig. 3 nanochannels detect Mir-31：Scatter diagram corresponding to (left side) typical single channel signal record (right side).
Nucleic acid samples different Fig. 4 and average residence time figure.
Fig. 5 nanochannels detect Mir-31：Scatter diagram corresponding to (left side) typical single channel signal record (right side).
Fig. 6 various concentrations Mir-31 current amplitude histogram and concentration curve.
Embodiment
Explanation that the present invention will be further explained by taking breast cancer as an example below.
Before introducing specific embodiment, the key instrument equipment used in the present invention and reagent are described below.
Capital equipment：Single probe ultra-low noise patch clamp amplifier：Axopatch 200B, MD companies of the U.S.（Axon）；
Axon Digidata1550B data collecting systems：1550B0/B1/B4, MD companies of the U.S.（Axon）；
Vibration isolators：TS/TM, Yiao Information Optical Science ＆ Technology Co., Ltd., Shanghai；
Supercentrifuge：JW-3021H, Anhui Jia Wen instrument and equipment Co., Ltd；
Electronic balance：BSA124S-CW, Beijing Sai Duolisi scientific instrument Co., Ltd；
Ultrapure water machine：Smart-N30uV, Shanghai Kanglei Analytical Instrument Co., Ltd.；
PH meter：UB-7, Beijing Sai Duolisi scientific instrument Co., Ltd.
Main agents：Lecithin：Sigma-Aldrich trade Co., Ltd lot number 850356-03-145；
Pentane：Sigma-Aldrich trade Co., Ltd lot number SZBD300BV；
Hexadecane：Sigma-Aldrich trade Co., Ltd lot number SHBD8135V；
Tris：Beijing Suo Laibao Science and Technology Ltd lot number 1120I0712；
α hemolysin：Sigma-Aldrich trade Co., Ltd lot number 124M4065V；
Potassium chloride：Tianjin Kermel Chemical Reagent Co., Ltd.'s lot number 20160305.
1st, the lookup and determination of breast Cancer Biomarkers thing
Searching the related MicroRNA marks of breast cancer has miR-31, miR-21, miR-200c, miR-145, miR-21,
MiR-147, miR-10b, miR-34a, miR-195, let-7a, miR-205, miR-373, miR-520.This experiment is chosen
MicroRNA marks miR-31 is detected.
2nd, based on detection of the nanometer single channel to breast Cancer Biomarkers thing
Experiment needs to use sample cell, and as Fig. 2 shows, sample cell is divided into two compartments of cis and trans（B, B'）, between compartment by
120-159 μm of Toflon films are kept apart.Hole A and hole A' be for first time liquid feeding body and insert electrode, afterwards liquid
The addition of body operates from following three aperture C and hole C'.Experimental procedure is as follows：
（1）Magneton is respectively put into chamber B and chamber B', then ultra-pure water is injected from hole A and hole A' respectively, with liquid level height
It is defined in micron openings, calculates the volume 2mL of required ultra-pure water；
（2）Magneton is taken out, pours out ultra-pure water, and rinses Toflon films, chamber and hole repeatedly, is finally rushed with absolute ethyl alcohol
Wash；
（3）After rinsing well, the drop remained in chamber and on Toflon films is dried up with nitrogen；
（4）Then, drip an about 10 μ L H liquid of drop respectively at micron openings on Toflon films both sides, and blow open rapidly, make
It extends on film scatters；
（5）Magneton is respectively put into chamber B and chamber B', then, 1mL is added with pipettor respectively from hole A and hole A' and contains 10
Mmol/L Tris, 1 mol/L KCl buffer solutions, then Ag/AgCl electrodes are inserted respectively；
（6）Then, hole A electrode is connected with the trans ends popped one's head in, and hole A' electrode is connected with the cis ends popped one's head in；
（7）The μ L L liquid of 1-2 drops about 15 is added dropwise in chamber B and chamber B' respectively with 25 μ L micropipettor, rapid featheriness, makes
L liquid is equably spread out on liquid level, then waits 30s-1min；
（8）Then, 1mL buffer solutions are added respectively from hole C and hole C', see whether to form phospholipid bilayer, such as formed,
Then carry out subsequent experimental；As do not formed, then blown and beaten repeatedly several times from hole C and hole C' ends, until it forms phospholipid bilayer；
（9）After phospholipid bilayer is formed, voltage+180mV is applied to system, alpha hemolysin its end of solution is added in the B' of hole
Concentration is 0.0125ng/mL, and open hole B' stirring system, controls suitable rotating speed, stir about 2min, forms nanometer single-pass
Road；
（10）After forming nanometer single channel, a certain amount of microRNA is added in the B' of hole, opens stirring system, stir about 5s
Afterwards, start to collect detection signal.
3rd, testing result and analysis
（1）As shown in Figure 3, Figure 4, successfully have detected Mir-31, base mispairing Mir-31 and composition Mir-31 probe
With Target, the results showed that either probe, Target or a base mispairing has different time and electricity with Mir-31
It can quickly and accurately be detected and be distinguished with label by stream, this method；
（2）As shown in figure 5, equally done distinguish Mir-31 it is preceding match somebody with somebody with it is rear with experiment, test result indicates that no matter probe with
Target front ends match or rear end matches, and because the end position for entering passage is different, its detection signal has two kinds of results, can
Made a distinction with Mir-31；
（3）As shown in fig. 6, successfully have detected the Mir-31 of various concentrations, least concentration can survey 0.01 nmol/ L, and make it
Concentration curve.
In summary, based on nanochannel 1 mol/ L KCl, 10 mmol/ L Tris buffer solution (pH 7.5) in
Markers for breast cancer Mir-31 is detected, has done contrast experiment, interference test etc. in experiment to it.Test result indicates that：This method
Successfully Mir-31 can be detected, and concentration gradient experiment has been done to it, minimal detectable concentration can reach 0.01 nmol/ L.
Claims (7)
1. a kind of method using nanochannel technology for detection cancer, this method needs to use a kind of sample cell, and sample cell is divided into
Two compartments of cis and trans（B, B'）, kept apart by 120-159 μm of Toflon films between compartment, hole A is with hole A'
For first time liquid feeding body and electrode is inserted, is connected by electrode with patch-clamp, chemical signal is converted into electric signal, afterwards
The addition of liquid operates from following three aperture hole C and hole C', it is characterised in that this method comprises the following steps：
（1）Magneton is respectively put into chamber B and chamber B', then injects ultra-pure water from hole A and hole A' respectively；
（2）Magneton is taken out, pours out ultra-pure water, and rinses Toflon films, chamber and hole repeatedly, is finally rushed with absolute ethyl alcohol
Wash；
（3）After rinsing well, the drop remained in chamber and on Toflon films is dried up with nitrogen；
（4）H liquid is separately added at micron openings on Toflon films both sides, and is blown open rapidly, it is extended on film and scatters；
（5）Magneton is respectively put into chamber B and chamber B', then, buffer solution is added with pipettor respectively from hole A and hole A',
Insert Ag/AgCl electrodes respectively again；
（6）Hole A electrode is connected with the trans ends popped one's head in, and hole A' electrode is connected with the cis ends popped one's head in；
（7）L liquid is added in chamber B and chamber B' respectively, rapid featheriness, L liquid is equably spread out on liquid level, then waits
30s-1min；
（8）Buffer solution is added respectively from hole C and hole C', sees whether to form phospholipid bilayer, is such as formed, then is carried out follow-up
Experiment；As do not formed, then blown and beaten repeatedly several times from hole C and hole C' ends, until it forms phospholipid bilayer；
（9）After phospholipid bilayer is formed, voltage is applied to system, alpha hemolysin solution, and open hole are added in the B' of hole
B' stirring system, suitable rotating speed is controlled, stir about 2min, forms nanochannel；
（10）After forming nanochannel, a certain amount of microRNA is added in the B' of hole, open stirring system, after stir about 5s,
Start to collect detection signal.
A kind of 2. method using nanochannel technology for detection cancer according to claim 1, it is characterised in that step
（1）Middle ultra-pure water addition is defined by liquid level higher than micron openings, calculates the volume 2mL of required ultra-pure water.
3. according to a kind of method using nanochannel technology for detection cancer described in claim 1 and 2, it is characterised in that step
Suddenly（4）The H liquid of middle addition is the mixed liquor that 10 ~ 15 μ L, H liquid are 1mL pentanes and 100 μ L hexadecanes.
4. according to a kind of method using nanochannel technology for detection cancer described in claim 1 and 2, it is characterised in that step
Suddenly（5）The buffer solution of middle addition includes 10mM Tris and 1M KCl, and addition is 0.8 ~ 1.1mL.
5. according to a kind of method using nanochannel technology for detection cancer described in claim 1 and 2, it is characterised in that step
Suddenly（7）The L liquid of middle addition is the mixed liquor that 15 ~ 25 μ L, L liquid are 25mg phosphatide and 2.5mL pentanes.
6. according to a kind of method using nanochannel technology for detection cancer described in claim 1 and 2, it is characterised in that step
Suddenly（8）The buffer solution of middle addition is 10mM Tris, and addition is 0.8 ~ 1.1mL.
7. according to a kind of method using nanochannel technology for detection cancer described in claim 1 and 2, it is characterised in that step
Suddenly（9）In to system apply voltage be+180V ~+200V, final concentration of 0.0125 ~ 0.145ng/mL of alpha hemolysin solution.
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|---|---|---|---|---|
| CN101680873A (en) * | 2007-04-04 | 2010-03-24 | 加利福尼亚大学董事会 | Use composition, equipment, the system and method for nano-pore |
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| CN203705389U (en) * | 2013-10-10 | 2014-07-09 | 华东理工大学 | Visualized microimaging nanochannel detection pool |
| CN103983487A (en) * | 2014-04-11 | 2014-08-13 | 西北大学 | Method for assembling alpha-hemolysin (alphaHL) protein nanopores by using single-layer phospholipid membrane |
| CN106319074A (en) * | 2016-10-17 | 2017-01-11 | 临沂大学 | LNA probe for detecting KRAS gene mutation and detection method |
-
2017
- 2017-07-21 CN CN201710599870.9A patent/CN107436317A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101680873A (en) * | 2007-04-04 | 2010-03-24 | 加利福尼亚大学董事会 | Use composition, equipment, the system and method for nano-pore |
| CN103298984A (en) * | 2010-02-08 | 2013-09-11 | 吉尼亚科技公司 | Systems and methods for manipulating a molecule in a nanopore |
| CN203705389U (en) * | 2013-10-10 | 2014-07-09 | 华东理工大学 | Visualized microimaging nanochannel detection pool |
| CN103983487A (en) * | 2014-04-11 | 2014-08-13 | 西北大学 | Method for assembling alpha-hemolysin (alphaHL) protein nanopores by using single-layer phospholipid membrane |
| CN106319074A (en) * | 2016-10-17 | 2017-01-11 | 临沂大学 | LNA probe for detecting KRAS gene mutation and detection method |
Non-Patent Citations (2)
| Title |
|---|
| CHAN CAO等: "Enhanced Resolution of Low Molecular Weight Poly(Ethylene Glycol)in Nanopore Analysis", 《ANALYTICAL CHEMISTRY》 * |
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