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Sample preparation method employing AFM (Atomic Force Microscope) for single antibody molecule imaging

An atomic force microscope and antibody molecule technology, applied in the nanometer field, can solve the problems of unsatisfactory imaging effect of single antibody molecule IgG and complicated preparation methods

Active Publication Date: 2016-06-22
SHANGHAI INST OF APPLIED PHYSICS - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The technical problem to be solved by the present invention is to overcome the problems in the prior art that the sample preparation method for atomic force microscope imaging of a single antibody molecule is relatively complicated, and the final imaging effect of a single antibody molecule IgG is not ideal, and a single antibody molecule is provided. Sample preparation method for molecular IgG atomic force microscopy imaging

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  • Sample preparation method employing AFM (Atomic Force Microscope) for single antibody molecule imaging
  • Sample preparation method employing AFM (Atomic Force Microscope) for single antibody molecule imaging
  • Sample preparation method employing AFM (Atomic Force Microscope) for single antibody molecule imaging

Examples

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Effect test

Embodiment 1

[0048] Example 1 Sample preparation for AFM imaging of a single digoxin antibody molecule IgG

[0049] 1) Mix single-stranded scaffold strand DNAM13mp18 and staple strand DNA coupled with digoxin in TAE-Mg at a molar ratio of 1:10 2+ Buffer system (40mM Tris-acetic acid, 1mM EDTA, 12.5mM MgCl 2 , pH 8.0), annealing from 95°C to 20°C with a PCR instrument at an annealing rate of 0.1°C / 10s to obtain an equilateral triangle DNA origami coupled with digoxin; the side length of the equilateral triangle DNA origami 130nm, with 3 digoxin antigens at equal intervals on each side, each digoxin antigen contains 2 digoxin molecules coupled to DNA origami with a distance of about 10-15nm; The nucleotide sequence of the staple strand DNA coupled with digoxin was published on March 17, 2010 in J.Am.Chem.Soc. Volume 132, Issue 10, Pages 3248-3249 entitled Goldnanoparticleself- The DNA sequence from A01 to Loop described on pages S11-S16 in the SupportingOnlineInformation of the paper simil...

Embodiment 2

[0053] Example 2 Sample preparation for AFM imaging of a single digoxigenin antibody molecule

[0054] 1) Mix single-stranded scaffold strand DNAM13mp18 and staple strand DNA coupled with digoxin in TAE-Mg at a molar ratio of 1:10 2+ The buffer system was annealed from 95°C to 20°C with a PCR instrument, and the annealing rate was 0.1°C / 10s, and a rectangular DNA origami coupled with digoxin was prepared; the side length of the rectangular DNA origami was 100nm×70nm. There are three digoxin antigens with equal spacing on each side, and each digoxin antigen contains two digoxin molecules coupled to DNA origami with a distance of about 8-13 nm; The nucleotide sequence of the staple-strand DNA with digoxin was published in the paper titled Self-Assembled Water-SolubleNucleicAcidProbeTilesforLabel-FreeRNAHybridizationAssays in Science Vol. 319 No. 5860 No. 180-183 on January 11, 2008 For the DNA sequences from 1 to 216 recorded on pages S15-S19 of the supplementary material, the ...

Embodiment 3

[0058] Example 3 AFM imaging of a single digoxin antibody molecule IgG

[0059] The sample of the atomic force microscope imaging of the single antibody molecule IgG prepared in Example 1 is subjected to AFM scanning imaging, and the scanning adopts a "J" scanning head, and the AFM probe is a silicon nitride probe (SNL, elastic coefficient 0.35N / m). The imaging mode of the atomic force microscope is the liquid phase "tapping" mode, select the local scanning range, control the imaging temperature at about 25°C, control the humidity at about 40%, control the scanning rate below 2Hz, and adjust the scanning parameters in the small force area (F< 200pN), the scan time is 1 minute.

[0060] Scanning results such as figure 1 As shown, the equilateral triangular DNA origami pattern of conjugated digoxigenin adsorbed on the mica surface is complete and clear, as figure 1 shown in a and 1b. The side length of the equilateral triangle is 130nm. A total of five antibody molecules Ig...

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Abstract

The invention discloses a sample preparation method employing an AFM (Atomic Force Microscope) for single antibody molecule imaging, a single antibody molecule IgG AFM imaging method and a DNA origami combined with antibody molecule IgG. The preparation method includes (1) mixing and reacting the DNA origami coupled with micromoleculeantigen at the edge with antibody of the micromoleculeantigen, wherein the micromoleculeantigen includes two identical micro molecules coupled to the edge of the DNA origami and spaced from each other by about 8-15 nanometers; (2) dropwise adding a reaction product on a newly-dissolved mica plate for adsorbing and imaging, and adding a buffer solution. Therefore, a sample is obtained. The preparation method provided by the invention is simple and convenient in operation. The antibody molecule IgG is absorbed on the mica surface with the aid of the DNA origami in a liquid environment, so that solid liquid interphasecapillary force effect on the antibody molecule IgG can be avoided and influence of a pinpoint effect on the antibody molecule IgG structure is reduced; and the imaging effect is improved.

Description

technical field [0001] The invention relates to the field of nanotechnology, in particular to a sample preparation method for single antibody molecule atomic force microscope imaging, an atomic force microscope imaging method for single antibody molecule IgG and a DNA origami combined with antibody molecule IgG. Background technique [0002] Atomic force microscopy (AFM) is one of the important tools for studying biomolecules at the nanoscale. Because of its ability to image biomolecules at the nanometer level under near-physiological conditions and obtain high-definition molecular morphology features, it has become an irreplaceable research method for studying the structure and function of biomolecules. [0003] Antibodies are an important class of biomolecules. They can neutralize toxins and viruses, promote phagocytosis by phagocytic cells, mediate immune cell activity, and stimulate complement, which play a very important role in the prevention, diagnosis, and treatment ...

Claims

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Application Information

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IPC IPC(8): G01Q30/20
CPCG01Q30/20
Inventor 周星飞赵志杰张萍李宾虞国凯胡钧郝长春杨家香
Owner SHANGHAI INST OF APPLIED PHYSICS - CHINESE ACAD OF SCI
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