A centrifugal microfluidic chip for extracting free nucleic acid and its method for extracting free nucleic acid

Abstract

The invention provides a centrifuging micro-fluidic control chip for free nucleic acid extraction and a method for extracting free nucleic acid, and belongs to the technical field of free nucleic acidseparating and extracting devices. The centrifuging micro-fluidic control chip comprises a disc, plasma separating chambers, nucleic acid extraction sample cavities, immiscible phase cavities and eluting cavities, wherein the plasma separating chambers, the nucleic acid extraction sample cavities, the immiscible phase cavities and the eluting cavities are in rotary symmetric distribution; each plasma separating chamber communicates with the corresponding nucleic acid extraction sample cavity through a corresponding passive siphon; and each nucleic acid extraction sample cavity communicates with the corresponding immiscible phase cavity and the corresponding eluting cavity through arch capillary microchannels. According to the centrifuging micro-fluidic control chip disclosed by the invention, a dynamic centrifuging micro-fluidic control method and a static immiscible phase micro-fluidic control method are combined for the first time, and the designed immiscible phase nucleic acid extracting and purifying structure realizes uniform mixing under the premise that the interface stability is not influenced; the plasma separating structure is integrated on the centrifuging micro-fluidiccontrol chip, so that the purpose of extracting and purifying free nucleic acid from 4mL whole blood fully-automatically within 15 minutes can be realized, the centrifuging micro-fluidic control chipis the fastest device for extracting and purifying the free nucleic acid from the whole blood at present, and the centrifuging micro-fluidic control chip is hopefully applied to liquid biopsy of thefree nucleic acid.

Description

technical field [0001] The invention belongs to the technical field of devices for separating and extracting free nucleic acids, and in particular relates to a centrifugal microfluidic chip for extracting free nucleic acids and a method for extracting free nucleic acids. Background technique [0002] "Liquid biopsy" is an important direction for tumor diagnosis and real-time monitoring. It has many advantages such as minimally invasive / non-invasive, real-time / multiple sampling, can overcome tumor heterogeneity, monitor tumor occurrence and development in real time, and guide individualized medication. clinical value. Free nucleic acid is a short nucleic acid fragment with a length of about 180 bp released by apoptotic cells, necrotic cells, and cell vesicles, and exists in blood, urine, cerebrospinal fluid, pleural effusion, saliva and other body fluids (Stroun M, Lyautey J, Lederrey C, et al. About the possible origin and mechanism of circulating DNA: Apoptosis and active ...

AI Technical Summary

Problems solved by technology

However, mixing is an essential step in nucleic acid extraction, which directly affects the efficiency and purity of extraction. However, most of the existing non-miscible static microfluidic nucleic acid extraction and purification methods use pipettes to achieve magnetic bead mixing, which has the risk of contamination. , and difficult to automate

[0004] Centrifugal microfluidic chips are one of the better technical means developed for on-site detection of nucleic acid POCT in recent years. Strohmeier proposed a centrifugal gas-phase transition magnetophoresis (GTM) for automatic realization of magnetic beads in The method of inter-chamber transfer, and it is used for nucleic acid extraction and purification. This method isolates the liquid in adjacent chambers through the "air gap", and realizes the transfer of magnetic beads with the help of external magnetic field force combined with centrifugal force. This method provides a method based on centrifugal micro Fluidic nucleic acid extraction and purification solution, but the maximum sample volume to be processed is 200uL, which may be because the chip is in a static state during the magnetic bead transfer process of this method, and the liquid in the adjacent chamber may be transferred through the magnetic beads under the action of gravity The mixing of channels will affect the purity of nucleic acid extraction. At the same time, the detection of free nucleic acid has a large demand for the original sample volume. Generally, the whole blood sample is not less than 3 ml, and the plasma sample is not less than 1.5 ml. For those relying on "air gap" Achieving liquid isolation between samples is somewhat difficult

[0005] Recently, Chi-Ju Kim (2018) et al. designed an integrated centrifugal microfluidic chip based on a solenoid valve, which can realize the integration of plasma separation and nucleic acid extraction and purification structure. The solenoid valve controls the opening and closing of the channel, making each functional chamber physically Space isolation, so that the extraction and purification of free nucleic acid from whole blood was realized on the microfluidic chip. This study shortened the pretreatment time of free nucleic acid to 30 minutes, but the extraction and purification process still included three washing steps, which may reduce the nucleic acid yield. Rate

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