CN101614647A - DNA liquid state test method based on atomic force microscope - Google Patents
DNA liquid state test method based on atomic force microscope Download PDFInfo
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- CN101614647A CN101614647A CN 200910055594 CN200910055594A CN101614647A CN 101614647 A CN101614647 A CN 101614647A CN 200910055594 CN200910055594 CN 200910055594 CN 200910055594 A CN200910055594 A CN 200910055594A CN 101614647 A CN101614647 A CN 101614647A
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Abstract
A kind of DNA liquid state test method based on atomic force microscope of technical field of biological comprises: configuration buffer solution, will wait that then observing the DNA sample solution dilutes, and makes the DNA sample solution; Get the DNA sample solution and drip on mica substrate, wait to adsorb the back, directly on mica substrate, be arranged to the picture detection cell at last with the ultrapure water flushing; Directly in the imaging detection cell, adopt atomic force microscope to carry out the scanning imagery processing after in the imaging detection cell, adding the imaging damping fluid.The present invention can be implemented in the AFM imaging of the dna molecular in the buffer solution that does not have the divalent ion existence, the observation of the bioprocesses that the DNA that needing in some buffer solution therefore can to avoid avoiding bivalent cation to participate in is relevant is for the conformation that detects dna molecular in the life process etc. provides effective means.
Description
Technical field
What the present invention relates to is a kind of detection method of technical field of biological, specifically is a kind of DNA liquid state test method based on atomic force microscope.
Background technology
Atomic force microscope (Atomic Force Microscopy has been released in the breadboard Christoph Gerber of C.F.Quate and the IBM Zurich cooperation of Binnig in 1985 and Stanford University, be called for short AFM), atomic force microscope is to realize high-resolution imaging by the interaction force between control and the test sample-needle point, it can obtain the material pattern on the nanoscale, while energy detection molecules interphase interaction power, and the electric conductivity to sample does not require, so it is widely used at life science.After AFM occurs, obtained using widely, be subjected to biologist's concern and favor at biological field.It can observe the architectural feature of biomacromolecule on single molecules level, can disclose interactional information between the single biomolecule simultaneously, can observe and understand the life dynamic process intuitively.These all are the targets that the biologist dreams of always.Therefore, various biomacromolecules are developed and are used as the atomic force microscope imaging technique of DNA, protein, RNA, enzyme, chromosome etc.
But, for biological sample, as DNA, RNA molecule etc., how with it at more firm fixing of substrate surface, can keep its original state again simultaneously, finally realize the unimolecule imaging, be the emphasis that many researchers pay close attention to always.For the preparation of atomic force microscope sample, the surface energy of the surface state of substrate and character such as substrate, surface charge and surface hydrophilic character etc. are for the crucial effect that all plays of fixing of final sample.And for the DNA sample, how therefore itself bear electricity, and the substrate mica that is generally used for the atomic force microscope imaging also is the bear electricity realize that the DNA sample fixedly is AFM imaging basic of realizing DNA on mica substrate.
The more method of report has following three kinds now: directly absorption method, electrostatic interaction fixation and covalence key are legal.But these method major parts all are confined to the dna molecular imaging under the atmosphere, and the relevant life process of a lot of DNA is only observed the truth that could reflect in the biosome in solution, thus develop simple possible, the AFM formation method that is used for the solution dna molecular reliably is the basis that utilizes the relevant life process of the atomic force microscope observation DNA biological respinse relevant with understanding DNA better.In sum, present stage is continued the AFM formation method of dna molecular in a kind of solution.
Summary of the invention
The present invention is directed to the prior art above shortcomings, a kind of DNA liquid state test method based on atomic force microscope is provided, be suitable for the imaging of AFM in solution and observe, and favorable reproducibility, good, the good stability of sharpness, can carry out repeatedly AFM scanning imagery; Can avoid simultaneously the observation of the biological respinse of the relative dna that bivalent cation participates in some buffer solution.
The present invention is achieved by the following technical solutions, the present invention includes following steps:
The first step, configuration buffer solution dilute dna solution to be observed then, make the DNA sample solution;
Described buffer solution is meant: concentration is the MgCl of 5.0 ~ 10.0mmol/L
2Solution;
Described dilution is meant: will wait to observe the DNA sample solution that DNA diluted sample to concentration is 2.5 ~ 5.0ng/ μ L;
Second step, get the DNA sample solution and drip on mica substrate, wait to adsorb the back, directly on mica substrate, be arranged to the picture detection cell at last with the ultrapure water flushing;
Described mica substrate is meant the fresh mica sheet of peeling off;
The resistance of described ultrapure water is greater than 18.2 megaohms;
Described imaging detection cell is meant that diameter is the polytetrafluoroethylene ring of 12mm;
The 3rd step, in the imaging detection cell, add the imaging damping fluid after, in the imaging damping fluid, carry out the atomic force microscope imaging and detect being fixed on DNA in the DNA sample solution on the mica substrate.
The 4-hydroxyethyl piperazine ethanesulfonic acid solution that described imaging damping fluid is a 40.0mmol/L concentration.
Described atomic force microscope uses the PicoScan type AFM instrument of U.S. MI company, adopts the small scanning head, and sweep limit is 6.5 * 6.5 μ m
2, imaging pattern is the MAC pattern, and the needle point of this atomic force microscope uses the MAClever type II type needle point of MI company, and resonant frequency is 78kHz, and force constant is 0.35N/m.Sweep velocity is controlled in the scope of 1.8-2.4 lines/s carries out.
Described imaging detects and is meant and the zone to be detected among the DNA is carried out 20-30 time atomic force scanning, DNA length, width and the conformation of acquisition correspondence position.
Fixed dna sample of the present invention, the preparation method is simple, easy controlled operation.The imaging reappearance of dna molecular, good stability.Each sample is fixed by this method of operating can both be at the afm image of the dna molecular under the solution that obtains better quality under the solution.Simultaneously, same sample area can not cause the drift and the damage of dna molecular through about 20-30 time scanning repeatedly.And, can be implemented in the AFM imaging of the dna molecular in the buffer solution that does not have the divalent ion existence by the method, the observation of the bioprocesses that the DNA that therefore can avoid some need avoid bivalent cation to participate in buffer solution is relevant is for the conformation that detects dna molecular in the life process etc. provides effective means.
Description of drawings
Fig. 1 is the AFM imaging of dna molecular under the atmosphere.
Fig. 2 is an embodiment imaging synoptic diagram.
Embodiment
Below embodiments of the invention are elaborated: present embodiment is being to implement under the prerequisite with the technical solution of the present invention, provided detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment.
Embodiment:
The first step, take by weighing the analytically pure magnesium chloride hexahydrate (MgCl of 203.0mg with electronic balance
2.6H
2O), dissolve in small beaker under the room temperature, go to constant volume in the 100.0mL volumetric flask then, making concentration is the magnesium chloride (MgCl of 10.0mmol/L
2) solution for standby, (concentration is: 0.5 μ g/ μ L) magnesium chloride solution with 10.0mmol/L is diluted to working concentration 5.0ng/ μ L (1.0mL), makes the DNA sample solution to get the original pUC18 DNA sample of 10 μ L;
Second step, get DNA sample solution 20.0 μ L and drip on the fresh mica sheet of peeling off, adsorbed 1 minute, wash with ultrapure water then, adding on moistening mica sheet directly under the situation about drying up without nitrogen that polytetrafluoroethylene ring that a diameter is 12mm is pressed on promptly forms a solution pool on the mica sheet, drip HEPES buffer solution 400.0 μ L then in solution pool.
The preparation process of described HEPES buffer solution is: the 4-hydroxyethyl piperazine ethanesulfonic acid that takes by weighing 953.24mg with electronic balance, in small beaker, dissolve under the room temperature, go to constant volume in the 100.0mL volumetric flask then, with NaOH modulation pH value is 7.5-8.0, and 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES) buffer solution that makes concentration and be 40.0mmol/L is preserved standby in 4 ℃ of refrigerators.
The 3rd goes on foot, detects in the liquid in buffering the DNA sample that is fixed on the mica sheet is carried out atomic force microscope imaging detection, 20-30 time atomic force scanning is carried out in zone to be detected among the DNA, and DNA length, width and the conformation of acquisition correspondence position are as shown in Figure 2.
Described atomic force microscope uses the PicoScan type AFM instrument of U.S. MI company, adopts the small scanning head, and sweep limit is 6.5 * 6.5 μ m
2, imaging pattern is the MAC pattern, and the needle point of this atomic force microscope uses the MAClever type II type needle point of MI company, and resonant frequency is 78kHz, and force constant is 0.35N/m.Sweep velocity is controlled in the scope of 1.8-2.4lines/s carries out.
Embodiment result shows: fixed dna sample of the present invention, the preparation method is simple, easy controlled operation.Each sample is fixed by this method of operating can both be at the afm image of the dna molecular under the solution that obtains better quality under the solution.Simultaneously, same sample area can not cause the drift and the damage of dna molecular through about 20-30 time scanning repeatedly.The imaging reappearance of dna molecular, good stability.And, can obtain the AFM imaging of single DNA molecules in the solution by the method, can reach nano level imaging resolution.
Claims (9)
1, a kind of DNA liquid state test method based on atomic force microscope is characterized in that, may further comprise the steps:
The first step, configuration buffer solution will wait that then observing the DNA sample solution dilutes, and makes the DNA sample solution;
Second step, get the DNA sample solution and drip on mica substrate, wait to adsorb the back, directly on mica substrate, be arranged to the picture detection cell at last with the ultrapure water flushing;
The 3rd step, in the imaging detection cell, add the imaging damping fluid after, in the imaging damping fluid, carry out the atomic force microscope imaging and detect being fixed on DNA in the DNA sample solution on the mica substrate.
2, the DNA liquid state test method based on atomic force microscope according to claim 1, it is characterized in that described buffer solution is meant: concentration is the MgCl of 5.0 ~ 10.0mmol/L
2Solution.
3, according to claim 1ly it is characterized in that based on DNA detection method in the solution of atomic force microscope solution described dilution is meant: DNA to be observed is diluted to the DNA sample solution that concentration is 2.5 ~ 5.0ng/ μ L.
4, the DNA liquid state test method based on atomic force microscope according to claim 1 is characterized in that, described mica substrate is meant the fresh mica sheet of peeling off.
5, the DNA liquid state test method based on atomic force microscope according to claim 1 is characterized in that the resistance of described ultrapure water is greater than 18.2 megaohms.
6, the DNA liquid state test method based on atomic force microscope according to claim 1 is characterized in that, described imaging detection cell is meant that diameter is the polytetrafluoroethylene ring of 12mm.
7, the DNA liquid state test method based on atomic force microscope according to claim 1 is characterized in that, the 4-hydroxyethyl piperazine ethanesulfonic acid solution that described imaging damping fluid is a 40.0mmol/L concentration.
8, the DNA liquid state test method based on atomic force microscope according to claim 1 is characterized in that, described atomic force microscope adopts the small scanning head, and sweep limit is 6.5 * 6.5 μ m
2, imaging pattern is the MAC pattern, and the resonant frequency of the needle point of this atomic force microscope is 78kHz, and force constant is 0.35N/m, and sweep velocity is controlled in the scope of 1.8-2.4 lines/s carries out.
9, the DNA liquid state test method based on atomic force microscope according to claim 1, it is characterized in that, described imaging detects and is meant and the zone to be detected among the DNA is carried out 20-30 time atomic force scanning, DNA length, width and the conformation of acquisition correspondence position.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8950011B2 (en) | 2013-05-22 | 2015-02-03 | International Business Machines Corporation | Targeted sequencing of biomolecules by pulling through a liquid-liquid interface with an atomic force microscope |
| CN107907450A (en) * | 2017-11-27 | 2018-04-13 | 深圳大学 | A kind of hydrophilic angle test method of two-dimension nano materials based on atomic force microscope |
| CN112684212A (en) * | 2020-12-26 | 2021-04-20 | 温州大学 | Liquid phase imaging method of atomic force microscope |
-
2009
- 2009-07-30 CN CN 200910055594 patent/CN101614647A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8950011B2 (en) | 2013-05-22 | 2015-02-03 | International Business Machines Corporation | Targeted sequencing of biomolecules by pulling through a liquid-liquid interface with an atomic force microscope |
| US8978161B2 (en) | 2013-05-22 | 2015-03-10 | International Business Machines Corporation | Targeted sequencing of biomolecules by pulling through a liquid-liquid interface with an atomic force microscope |
| US9689034B2 (en) | 2013-05-22 | 2017-06-27 | International Business Machines Corporation | Targeted sequencing of biomolecules by pulling through a liquid-liquid interface with an atomic force microscope |
| US10041114B2 (en) | 2013-05-22 | 2018-08-07 | International Business Machines Corporation | Targeted sequencing of biomolecules by pulling through a liquid-liquid interface with an atomic force microscope |
| CN107907450A (en) * | 2017-11-27 | 2018-04-13 | 深圳大学 | A kind of hydrophilic angle test method of two-dimension nano materials based on atomic force microscope |
| CN107907450B (en) * | 2017-11-27 | 2020-06-09 | 深圳大学 | Two-dimensional nano material hydrophilic angle testing method based on atomic force microscope |
| CN112684212A (en) * | 2020-12-26 | 2021-04-20 | 温州大学 | Liquid phase imaging method of atomic force microscope |
| CN112684212B (en) * | 2020-12-26 | 2022-08-30 | 温州大学 | Liquid phase imaging method of atomic force microscope |
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