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Method for preparing bio-macromolecular monomolecular chips by virtue of high-density nano-dot arrays

A technology of biological macromolecules and nano-dot arrays is applied in the field of fabrication of high-density single-molecule chips of biological macromolecules to achieve the effect of simple fabrication process.

Active Publication Date: 2015-04-22
SHANGHAI BEION MEDICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the DNA entering the array after rolling circle replication is not a single-copy fragment of the natural genome, but multiple copies of short fragments, that is, the chip is not a true single-molecule DNA chip

Method used

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  • Method for preparing bio-macromolecular monomolecular chips by virtue of high-density nano-dot arrays

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] In the first step, a photoresist PMMA with a thickness of 10 nm was spin-coated on the silicon substrate after cleaning with Piranha solution and deionized water.

[0027] In the second step, a 30nm nanopit pattern was developed on the negative film by electron beam lithography (the diameter of the nanodot is controlled to accommodate only one nucleic acid loading tool), and the spacing of the nanopit is controlled at 500nm by mask design.

[0028] In the third step, the developed substrate is treated with plasma to hydroxylate the silicon surface; the substrate is washed with acetone to remove the PMMA film, and an array of hydroxylated nano-dots is obtained.

[0029] In the fourth step, the substrate is treated with an APTES solution to aminate the surface of the nano-dots, and after cleaning, it is treated with Biotin-NHS to selectively biotinylate the surface of the nano-dots.

[0030] The fifth step is to immobilize the tetramer anti-streptavidin-single DNA complex...

Embodiment 2

[0033] In the first step, the photoresist PMMA / MA with a thickness of 10nm is spin-coated on the existing glass mica substrate, so that it does not exceed the diameter of a subsequent single molecule.

[0034] In the second step, electron beam lithography is used and developed to realize a nano-dot array with a diameter of less than 20nm, and the spacing of the nano-pit is controlled at 200nm by mask plate design.

[0035] In the third step, the developed substrate is coated with O 2 The surface of the substrate was hydroxylated by plasma treatment; the substrate was washed with acetone to remove the PMMA film, and an array of hydroxylated nano-dots was obtained.

[0036] In the fourth step, the substrate is treated with an APTES solution to aminate the surface of the nano-dots, and after cleaning, it is treated with Biotin-NHS to selectively biotinylate the surface of the nano-dots.

[0037] The fifth step is to immobilize the tetrameric anti-streptavidin-single RNA complex ...

Embodiment 3

[0040] In the first step, a photoresist LIGA film with a thickness of 20 nm was spin-coated on the cleaned glass substrate.

[0041] In the second step, a pattern with a diameter of 20nm is developed on the film lithography by electron beam lithography technology, and the nanopit spacing is controlled at 500nm by mask plate design.

[0042] In the third step, the developed substrate is coated with O 2 The surface of the substrate was hydroxylated by plasma treatment, and the substrate was washed with acetone to remove the PMMA film to obtain a hydroxylated nanodot array.

[0043] In the fourth step, the substrate is treated with an APTES solution to aminate the surface of the nano-dots, and after cleaning, it is treated with Biotin-NHS to selectively biotinylate the surface of the nano-dots.

[0044] In the fifth step, the TNF-α monoclonal antibody complex bound to TNF-α (tumor necrosis factor) and labeled with biotin is immobilized on the surface of the nanodots. The diameter ...

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Abstract

The invention discloses a method for preparing bio-macromolecular monomolecular chips by virtue of high-density nano-dot arrays. The method comprises the following steps: firstly, laying a layer of thin films on a substrate, then preparing nano-hollow arrays on the thin films, performing further substrate modification (performing hydroxylation on the substrate which is subjected to O2 plasma treatment, and then modifying the substrate to be biotinylated), and removing the thin films to form active nano-dot arrays which can be connected with bio-macromolecules, wherein only one bio-macromolecule can be connected with each nano-dot because the nano-dots are small enough; and finally cleaning the substrate to obtain high-density monomolecular biological chips. Objective biological chips obtained by using the method disclosed by the invention are mainly characterized in that the objective biological chips are monomolecular, have high throughput and multiple functions, and can be applied to the fields of ultra-sensitive monomolecular enzyme-linked immunosorbent assay, hybridization based DNA variation detection, monomolecular DNA synthesis and connection sequencing, single-cell RNA sequencing and the like.

Description

technical field [0001] The invention relates to a biochip manufacturing technology, in particular to a method for manufacturing a biomacromolecule (namely DNA, RNA and protein) high-density single-molecule chip. Background technique [0002] High-density biomacromolecule (DNA, RNA, and protein) single-molecule chips, that is, each sample point has only one biomacromolecule, and the distance between sample points reaches the limit of optical resolution, which can maximize the amount of data per unit area; The single molecule can be modified by molecular biological means and used as "bait" to capture the target molecule, which can be used for the interaction between molecules such as antigen and antibody, DNA hybridization detection of single nucleotide polymorphism, copy number variation And comparative genome hybridization chips, etc., can also be used for DNA synthesis sequencing, ligation sequencing, etc., to convert the second-generation sequencing technology into the thi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G01N33/68C40B50/18C40B40/10C40B40/06
Inventor 王志民程秀兰吕鹏雨杨秋萍王跃何珊珊
Owner SHANGHAI BEION MEDICAL TECH CO LTD
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