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Nano-structure-based protein unimolecular electronic device and preparation and application thereof

A technology for electronic devices and proteins, which is used in biochemical equipment and methods, and the determination/inspection of microorganisms. Single-molecule sequencing, improving capture efficiency, and achieving the effect of continuous sequencing

Inactive Publication Date: 2017-07-07
PEKING UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The second-generation sequencing technology has the following disadvantages: (1) DNA sequencing library preparation requires a large amount of initial DNA samples; (2) parallel amplification of samples may lead to bias in sequencing library preparation; (3) preparation of sequencing libraries requires a large number of (4) base-by-base reading is performed on the DNA sequencing template through the DNA biosynthesis reaction, and only one base can be read in one biochemical reaction, and the read length is relatively short. It is also the bottleneck that makes it difficult to increase the sequencing speed and throughput; (5) The reporting cycle of sequencing results is long
The present invention is aimed at the problems of high cost, low accuracy and poor repeatability in current single-molecule DNA sequencing, by detecting the fluctuation of the conductivity of protein molecules such as DNA polymerase or RNA polymerase in the nucleic acid synthesis process, inferring single-stranded nucleic Develop a low-cost, rapid nucleic acid sequencing device

Method used

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  • Nano-structure-based protein unimolecular electronic device and preparation and application thereof
  • Nano-structure-based protein unimolecular electronic device and preparation and application thereof
  • Nano-structure-based protein unimolecular electronic device and preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Embodiment 1: DNA polymerase molecular electronic device based on nanopore-counter electrode structure

[0051] (1) Preparation of nanopore-counter electrode structure: use a series of micro-nano processing methods such as photolithography, dry etching, wet etching, reactive ion beam etching, etc. 2 / SiN substrate is processed to prepare a suspended membrane structure 1 with a SiN film with a size of 2 square microns and a thickness of 30 nanometers, such as figure 1 shown. Among them, SiO 2 The film 1b acts as an insulating buffer layer to support the SiN film 1c and reduce the capacitive effect in electrical measurement; the Si substrate 1a mainly supports the SiO 2 The role of membrane 1b. The material of the suspended membrane structure 1 is not limited to Si / SiO 2 / SiN, and other materials such as graphene can also be used to prepare a suspended membrane structure. On the suspended film structure 1, the nano-counter electrode 3 is prepared by a conventional el...

Embodiment 2

[0058] Embodiment 2: RNA polymerase molecular electronic device based on nanopore-counter electrode structure

[0059] (1) Preparation of nanopore-counter electrode structure: Same as Example 1.

[0060] (2) The modification of the surface of the nano-pair electrode: the same as in Example 1.

[0061] (3) Preparation of nanopore-counter electrode-protein composite structure by chemical crosslinking method: inject 500 microliters of 1:1 mixed solution of NHS solution (100mM) and EDC solution (100mM) into the centrifuge tube, put For the chemically modified nanopore-counter electrode chip, 200 microliters of RNA polymerase (50 micrograms / ml) was dropped into a centrifuge tube, and stored in the dark for 1 hour, so that the RNA polymerase and the nanometer counter electrode were cross-linked. After the end, take it out, soak it in deionized water, and save it for later use. The nanopore-counter electrode-protein composite structure has the function of detecting DNA sequence.

...

Embodiment 3

[0066] Embodiment 3: DNA exonuclease molecular electronic device based on nanopore-counter electrode structure

[0067] (1) Preparation of nanopore-counter electrode structure: Same as Example 1.

[0068] (2) The modification of the surface of the nano-pair electrode: the same as in Example 1.

[0069] (3) Preparation of nanopore-counter electrode-protein composite structure by chemical crosslinking method: inject 500 microliters of 1:1 (volume ratio) mixed solution of NHS solution (100mM) and EDC solution (100mM) into the centrifuge tube, put into the chemically modified nanopore-counter electrode chip, drop 200 microliters of DNA exonuclease (50 micrograms / ml) into a centrifuge tube, and store it in the dark for 1 hour, so that the DNA exonuclease and the chemically modified nano Gold cross-links the electrodes. After the end, take it out, soak it in deionized water, and save it for later use. The nanopore-counter electrode-protein composite structure has the function of ...

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Abstract

The invention discloses a nano-structure-based protein unimolecular electronic device and preparation and application thereof; single protein or its complex molecules are fixed to a counter electrode at a nano hole, dynamical features of conformational fluctuation of the protein are characterized by detecting the electrical conductivity of the protein or its complex molecules in a solution, and the activity of the protein and the biochemical reactive process thereof with substrate molecules are detected. The nano-structure-based protein unimolecular electronic device may act as a third-generation sequencing method having great development potential, preparing special DNA sequencing libraries is not required, labeling nucleic acids is not required, ultra-long, continuous, quick and precise base reading can be performed, and ultralow-cost DNA sequencing and otherness can be achieved if a high-throughput molecular device is prepared.

Description

technical field [0001] The invention relates to a single-enzyme biosensor, which can be used for the sequencing of single-molecule DNA, and can also be used for detecting substrate molecules with extremely low concentration in solution, and belongs to the third-generation DNA sequencing device. Background technique [0002] DNA sequencing technology is one of the important milestones in the development of modern life sciences. In the past ten years, the emergence of low-cost and high-throughput DNA sequencing technology has caused the explosive growth of the largest and most core nucleic acid sequence information in the highly complex system of life, which has brought unprecedented opportunities and challenges to life science and medicine. . The thorough deciphering of the genetic code of life will become possible, and the popularization of big data on genetic information will benefit the survival and health of every ordinary member of human society. [0003] The next-gene...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6869C12Q2565/607
Inventor 陆祖宏李清宁丁韬力严勇万成魏颖颖孙利李成强孙杰王磊
Owner PEKING UNIV
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