DNA nanostructure-modified microfluidic chip for optical biosensing and preparation and application of DNA nanostructure-modified microfluidic chip

A microfluidic chip and nanostructure technology, applied in biochemical equipment and methods, bioreactors/fermenters for specific purposes, microorganisms, etc., can solve the problems of cumbersome operation and low detection efficiency of circulating tumor cells, and achieve high sensitivity High, specific, and easy-to-operate effects

Active Publication Date: 2019-10-08
SHANGHAI ADVANCED RES INST CHINESE ACADEMY OF SCI
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

[0009] In view of the shortcomings of the prior art described above, the purpose of the present invention is to provide a microfluidic chip modified with a DNA nanostructure for optical biosensing and its preparation and application as well as its preparation method and use, for solving existing problems. In the technology, the detection efficiency of circulating tumor cells is low and the operation is cumbersome.

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  • DNA nanostructure-modified microfluidic chip for optical biosensing and preparation and application of DNA nanostructure-modified microfluidic chip
  • DNA nanostructure-modified microfluidic chip for optical biosensing and preparation and application of DNA nanostructure-modified microfluidic chip
  • DNA nanostructure-modified microfluidic chip for optical biosensing and preparation and application of DNA nanostructure-modified microfluidic chip

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Embodiment 1

[0070] (1) Materials and equipment

[0071]The electrophoresis devices were all purchased from Bio-Rad; the PCR instrument was Peltier thermal cycler PTC-200 (MJ.Research Inc., SA); the UV gel instrument was G:Box (gene Company Limited); -3010); fluorescence spectrophotometer (Hitachi F-4500); HD-850 desktop horizontal flow clean bench purchased from Shanghai Sujing Industrial Co., Ltd.; fluorescence confocal microscope (Leical, SP8); total internal reflection fluorescence microscope (Leical); inverted fluorescence microscope (Leical); flow cytometry (BD array).

[0072] (2) Construction of microfluidic chip system modified by DNA tetrahedron

[0073] Construction ideas: The microfluidic chip is divided into upper and lower layers. The lower layer of the chip is decorated with a DNA tetrahedral structure. The DNA tetrahedral structure can be combined with the HCR hybridization chain reaction to achieve signal amplification. The initiator chain in the HCR structure can be hybr...

Embodiment 2

[0083] Example 2 Detection limit and linear range of microfluidic chip modified by DNA tetrahedron

[0084] Different numbers of MCF 7 cells (10, 100, 1000, 10000, 100000) were captured using the aptamer-HCR-DNA tetrahedron structure prepared in Example 1 and the fishbone microfluidic chip. Add different amounts of cells into the probe solution, incubate with shaking on ice for 30 min, wash with BB three times, calculate the capture rate respectively, and obtain the detection limit and linear range. The specific calculation method of the capture rate is: the cells combined with the HCR-aptamer probe are incubated and washed, which is called the stock solution; the same cells combined with the HCR-aptamer probe are incubated and washed, and then passed into the microfluidic channel to collect the captured liquid and become the recovery solution , use a fluorescence microscope to observe and count the cells in the original solution and the recovered solution, and the amount of c...

Embodiment 3

[0086] Example 3 DNA Tetrahedral Modified Microfluidic Chip Detection of Purity

[0087] Mix 1 mL of the two types of cells (MCF7, Hela) in equal amounts, add to the probe solution, incubate with shaking on ice for 30 min, and wash with BB three times. Under different cell numbers (100, 1000, 10000), the optimized aptamer-HCR-DNA tetrahedral structure probe in Example 1 and the channel of the fishbone microfluidic chip were used to capture and calculate the capture purity. The specific calculation method of purity is: MCF7 / Hela cells combined with HCR-aptamer probes are incubated and washed, which is called stock solution; the same MCF7 / Hela cells combined with HCR-aptamer probes are incubated and washed, and then passed into the microfluidic channel to collect and capture The final liquid becomes the recovery solution. Use a fluorescence microscope to observe and calculate the number of MCF7 cells in the original solution and the recovery solution. The amount of captured cell...

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Abstract

The invention provides a DNA nanostructure-modified microfluidic chip for optical biosensing and preparation and application of the DNA nanostructure-modified microfluidic chip. A system includes a microfluidic channel structure and a (hybridization chain reaction) HCR structure connected with an Aptamer. The microfluidic channel structure includes an upper layer and a lower layer, wherein the upper layer is a PDMS material plate, the lower layer is a microarray substrate, a reaction cavity is formed in the PDMS material plate, and the surface of the microarray substrate is modified with a DNAtetrahedron. The DNA tetrahedron is formed by four nucleotide single chains of A, B, C and D, wherein the A chain can be complementarily connected with a H2 chain base in the HCR structure in a paired mode, and the HCR structure includes an initiation chain I and a stem loop, namely, a H1 chain and a H2 chain, the 5' ends of the H1 chain and the H2 chain are modified with fluorescent groups, andthe initiation chain I is connected with the Aptamer. By adopting the system, the purpose of capturing circulating tumor cells efficiently is achieved.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a microfluidic chip modified with a DNA nanostructure for optical biosensing and its preparation and application as well as its preparation method and use. Background technique [0002] Malignant tumors are a disease with high mortality both at home and abroad, and their adverse effects on social and economic development are becoming more and more significant. At present, the traditional methods of tumor diagnosis and monitoring mainly include imaging examination, biopsy and traditional blood tumor marker detection. However, imaging examinations have high requirements for operators, and usually only have a good ability to distinguish tumors larger than 1cm; cytology or histology biopsy is invasive, resulting in low patient acceptance; traditional blood tumors Due to the lack of reliable markers in marker detection, the accuracy of the results is not high. [0003] Circulating tumor...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12M1/00C12N5/09
CPCC12M23/16C12M47/04C12N5/0693C12N2509/10
Inventor 宓现强王晨光徐怡
Owner SHANGHAI ADVANCED RES INST CHINESE ACADEMY OF SCI
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