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PCR chip based on droplet array and application thereof

An array and chip technology, applied in the field of real-time quantitative PCR chip and its fluorescence detection system, can solve the problems of water evaporation, fusion and translocation of droplets, etc., and achieve the effect of avoiding interference and reducing mutual pollution

Active Publication Date: 2011-08-31
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this system has not been used in the qPCR system, which may be due to the inevitable water evaporation during the droplet generation process and the easy fusion and translocation of droplets during the PCR thermal cycle process.

Method used

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  • PCR chip based on droplet array and application thereof
  • PCR chip based on droplet array and application thereof
  • PCR chip based on droplet array and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Prepare chip:

[0041] A 6-inch monocrystalline silicon wafer is oxidized in a high-temperature furnace at 1100°C for 2 hours, and SiO is formed on the surface 2 oxide layer, and then placed in 1% octadecyltrichlorosilane (solvent is toluene) solution at room temperature for 2 hours to make SiO 2 The surface of the oxide layer is silanized, and after taking it out, wash it with toluene, isopropanol and absolute ethanol, and finally wash it with deionized water and dry it in the air. Next, put the silicon wafer in the glue-spinning machine, drop a small amount of AZ photoresist on the surface, spin the glue at 2000rpm for 1 minute, and then put the silicon wafer in the glue-baking machine at 90°C to heat the film for 10 minutes and cool it down. Finally, a mask printed with light-transmitting array dots is covered on the surface of the photoresist. The light-transmitting array dots on the mask are circular dots with a diameter of 300um, forming a 6×6 square array. The m...

Embodiment 2

[0044] Embodiment 2 Multi-step addition reagent stability and reproducibility investigation

[0045] Take the fluorescein dye solution as the sample, use a pipette with a range of 0.1-2.5 μL, add the sample three times in sequence on the 6×6 array chip, and generate 100 nL for the first time with a concentration of 10 -6 mol / L fluorescein solution droplet array (as attached image 3 a), then add 100nL concentration of 10 to the generated droplets -5 mol / L fluorescein solution (as attached image 3 b), finally add 300nL concentration of 10 -5 mol / L fluorescein solution (as attached image 3 shown in c). attached image 3 Among them, the upper row is a bright field photo (directly photographed with a common tungsten light source), and the lower row is a fluorescence photo. The fluorescence intensity was analyzed with a data processing program. According to the average value of the fluorescence intensity of 36 droplets, the relative standard deviation can be calculated to b...

Embodiment 3

[0046] Example 3 microRNA quantitative analysis

[0047] For most RT-PCR reaction systems using RNA as a template, reverse transcription reaction and PCR amplification need to be operated separately in two steps. In order to demonstrate the applicability of the system for two-step real-time quantitative RT-PCR detection and analysis, the detection sensitivity and linear range of the system were investigated using artificially synthesized mir-122 as a sample. First, generate 6×6 100nL droplets on the chip (chip array with attached image 3 Consistent), each column of 6 droplets contains the same concentration of mir-122 samples, the amount of mir-122 added to the 6 columns of droplets is (from right to left) blank, 9.6×10 3 , 9.6×10 4 , 9.6×10 5 , 9.6×10 6 , 9.6×10 7 copies, spanning 5 orders of magnitude. Next, add 100nL reverse transcription reagent master mix solution (reverse transcription master mix solution volume is 10uL, contains dNTP mixed solution concentration ...

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Abstract

The invention discloses a PCR (Polymerase Chain Reaction) chip based on droplet array. The chip is obtained by taking a mono crystalline silicon sheet or a glass sheet as a base material and adopting standard photoetching as well as wet etching technique. The mono crystalline silicon sheet or the glass sheet is the base material of the chip. A SiO2 oxide layer and a silane layer generated by silanization on the surface of the SiO2 oxide layer are adhered to the base material successively. A hydrophilic pit array, which can be a droplet array and is formed by etching with photoresist technology, is disposed on the silane layer. A closed circular guardrail is arranged on the outer periphery of the hydrophilic pit array and the guardrail is connected with the silane layer in a sealed manner. The invention also discloses the application of the chip on real-time quantitative PCR detection and real-time quantitative isothermal amplification reaction detection. The detection system provided in the invention remarkably reduces the reaction volume and the detection sensitivity of the system fairly equals that of a commercialized quantitative PCR instrument. The detection system in the invention is easy for operation and has low cost, thus can be applied to commercialization.

Description

1. Technical field: [0001] The invention relates to a real-time quantitative PCR chip and a fluorescence detection system thereof, in particular to a droplet array-based PCR chip and a fluorescence imaging detection system. 2. Background technology: [0002] Real-time quantitative (reverse transcription) polymerase chain reaction (hereinafter referred to as qPCR or qRT-PCR) technology is based on traditional PCR technology, which can accurately quantify the copy number of original nucleic acid (including DNA and RNA) templates. A modern molecular biology detection technique for analysis. Due to the advantages of high accuracy and wide linear range, qPCR technology has been widely used in molecular diagnosis, disease research, clinical medicine and other fields. qPCR technology usually follows the amplification principle of traditional PCR, the difference is that real-time quantitative detection is performed in the annealing or extension stage of each cycle, and the logarith...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12M1/00C12M1/34C12Q1/68G01N21/64
Inventor 姚波方群张云霞祝莹
Owner ZHEJIANG UNIV
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