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Single cell rt-pcr chip and preparation method thereof

A RT-PCR and single-cell technology, applied in the field of single-cell research, can solve problems such as complex structures, reduced single-cell occupancy, and difficulty in liquid entry, achieving high throughput and high single-cell occupancy

Active Publication Date: 2021-06-08
INST OF SEMICONDUCTORS - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

If the volume of the microwell is increased, multiple cells may appear in one microwell. Even if the multicellular situation can be reduced by diluting the cell suspension, this Poisson distribution greatly reduces the utilization of the microwell.
[0006] The water-in-oil microfluidic chip uses a T-shaped structure to produce a water-in-oil system. In this method, the single-cell occupancy rate is still limited to the Poisson distribution. Increasing the droplet volume greatly reduces the single-cell occupancy rate. Due to the microfluidic sealing structure, it is difficult to enter the liquid in the later stage
[0007] The microfluidic chip with fine structural design can control the liquid in multiple steps through the switch control of the valve. Although this type of chip has been successfully applied to single-cell research, its complex structure makes it costly and difficult to operate.

Method used

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  • Single cell rt-pcr chip and preparation method thereof
  • Single cell rt-pcr chip and preparation method thereof
  • Single cell rt-pcr chip and preparation method thereof

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preparation example Construction

[0075] According to the general inventive concept of the present invention, as another aspect of the present invention, the present invention also provides a method for preparing a single-cell RT-PCR chip, comprising the following steps:

[0076] Step 1: Take a substrate and clean the substrate to remove organic impurities and inorganic impurities on the surface;

[0077] Step 2: Prepare a double-set microarray structure on the substrate to obtain a base: 20 with a double-set microarray structure. The array arrangement form of the double-set microarray includes rectangle, ring, mirror image and close arrangement;

[0078] Step 3: performing surface treatment on the substrate 20;

[0079] Step 4: Surround the double-hole microarray structure inside with a sealing ring 29 , and press the sealing ring with a cover sheet 28 to complete chip packaging.

[0080] Further, the preparation process of the double-well microarray is as follows:

[0081] Step 21: uniformly spin-coat a la...

Embodiment 1

[0108] This embodiment provides a single-cell RT-PCR chip with a double-well microarray structure, such as Figure 4 As shown, the array of the microarray of double sets of holes of the chip is arranged in a rectangle, and the double sets of holes include a first hole 30 and a second hole 31. The preparation process of the chip under working conditions is as follows: figure 1 As mentioned, the details are as follows:

[0109] (1) Take a substrate, which is a non-biotoxic material;

[0110] Preferably, the non-biologically toxic materials include quartz glass, silicon wafers, silicon dioxide wafers, optical fiber panels, organic thin sheets, and medical stainless steel;

[0111] Preferably, the substrate is cleaned to remove organic impurities and inorganic impurities on the surface;

[0112] More preferably, the cleaning method of the substrate includes organic cleaning and inorganic cleaning; wherein, the organic cleaning process is: sequentially using acetone, ethanol, dei...

Embodiment 2

[0146] This embodiment provides a single-cell RT-PCR chip with a double-well microarray structure, such as Figure 5 As shown, the array of the microarray of the double set of holes of the chip is arranged in a rectangle, and the double set of holes includes a first hole 30 and 5 second holes 31. The preparation process of the chip under working conditions is as follows: figure 1 As mentioned, the details are as follows:

[0147] Step 1: Take a substrate, which is a non-biotoxic material;

[0148] Preferably, the non-biologically toxic materials include quartz glass, silicon wafers, silicon dioxide wafers, optical fiber panels, organic thin sheets, and medical stainless steel;

[0149] Preferably, the substrate is cleaned to remove organic impurities and inorganic impurities on the surface;

[0150] More preferably, the cleaning method of the substrate includes organic cleaning and inorganic cleaning; wherein, the organic cleaning process is: sequentially use acetone, ethano...

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Abstract

The single-cell RT-PCR chip provided by the present invention includes a substrate with a double-set hole microarray structure; the double-set hole microarray structure includes several double sets of holes, and the double set of holes includes first holes and The second hole at the inner bottom; the second hole realizes single cell capture, and the first hole can hold the reaction solution as a RT-PCR reaction pool. The single-cell RT-PCR chip provided by the present invention solves the problem of small volume of the reaction pool in the traditional single-cell microwell array, and realizes single-cell capture and reaction reagent injection efficiently and quickly; at the same time, the chip of the present invention has a simple structure and does not require complicated The microfluidic liquid circuit saves costs and provides the greatest possibility for the commercialization of single-cell RT-PCR.

Description

technical field [0001] The invention belongs to the field of single cell research, in particular to a single cell PT-PCR chip and a preparation method thereof. Background technique [0002] In 1985, Kary.B.Mullis, a scientist in the Human Genetics Laboratory of Cetus Corporation in the United States, invented the epoch-making polymerase chain reaction (Polymerase Chain Reaction, PCR) under the inspiration of great inspiration, which realized unlimited amplification in vitro DNA fragments dream. Subsequently, Saiki successfully amplified human β-globin DNA by PCR method for the first time, and applied it to the prenatal diagnosis of sickle cell anemia. [0003] PCR is a method of in vitro enzymatic reaction to synthesize specific DNA. It utilizes DNA polymerase reaction mediated by artificial synthetic primers to amplify a DNA fragment located between two known sequences. Single-cell reverse transcription-polymerase chain reaction (Reverse Transcription-Polymerase Chain Rea...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12M1/00
Inventor 王琛瑜俞育德刘文文杨翎魏清泉
Owner INST OF SEMICONDUCTORS - CHINESE ACAD OF SCI