CN110016435B - A centrifugal microfluidic chip for free nucleic acid extraction and method for extracting free nucleic acid - Google Patents

Abstract

The invention provides a centrifugal microfluidic chip for free nucleic acid extraction and a method for extracting free nucleic acid, belonging to the technical field of free nucleic acid separation and extraction devices. The centrifugal microfluidic chip includes a disc and a rotationally symmetrical distribution of plasma separation chamber, nucleic acid extraction sample chamber, immiscible phase chamber and elution chamber; the plasma separation chamber and the nucleic acid extraction sample chamber are connected by a passive siphon, and the nucleic acid extraction sample chamber , the immiscible phase cavity and the elution cavity are connected by arched capillary microchannels. The invention combines the dynamic centrifugal microfluidic method with the static immiscible phase microfluidic method for the first time, and the designed immiscible phase nucleic acid extraction and purification structure realizes mixing without affecting the interface stability; the plasma separation structure is integrated into On the centrifugal microfluidic chip, it can automatically extract and purify free nucleic acid from 4 mL of whole blood within 15 minutes. It is the fastest device for extracting and purifying free nucleic acid from whole blood.

Description

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

technical field

The invention belongs to the technical field of free nucleic acid separation and extraction devices, and in particular relates to a centrifugal microfluidic chip for free nucleic acid extraction and a method for extracting free nucleic acid.

Background technique

"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, overcoming tumor heterogeneity, real-time monitoring of tumor occurrence and development, and guiding individualized medicine. of clinical value. Free nucleic acid is a short nucleic acid fragment of about 180 bp in length released by apoptotic cells, necrotic cells and cell vesicles, and exists in blood, urine, cerebrospinal fluid, pleural effusion, saliva and other body fluids (Strour M, Lyutey J, Lederrey C, et al.About the possible origin and mechanism of circulating DNA:Apoptosisand active DNA release[J].Clinica Chimica Acta,2001,313(1-2):139-142.), is an important molecular marker of "liquid biopsy" source. In cancer patients, a part of cell-free nucleic acid originates from the tumor, called circulating tumor DNA (ctDNA), which has the same mutation and genetic changes as the primary tumor. By detecting these specific nucleic acid fragments, early cancer screening, Cancer status detection and prognosis review and other research applications (Crowley E, Di N F, Loupakis F, et al. Liquid biopsy: monitoring cancer-genetics in theblood. Nat RevClin Oncol [J]. Nature Reviews Clinical Oncology, 2013, 10 (8):472.). There is also a type of cell-free DNA (cfDNA), which is your own DNA that is free in the blood. However, the content of both cfDNA and ctDNA in whole blood samples is extremely low, with an average content of about 10-30 ng/mL; it is difficult to analyze directly, and multiple pre-processing steps including plasma separation, nucleic acid extraction and purification are required. The time interval used, storage conditions, and careful pretreatment can all affect the concentration of cfDNA or ctDNA. Another problem is that short fragments of cfDNA or ctDNA are both less stable, with a half-life of about 4-30 minutes in vivo and a half-life of about 1.5 to 2 hours after blood draw. At present, whole blood is mainly stored commercially by means of special blood-free nucleic acid storage tubes to ensure that the free nucleic acid itself will not be degraded within a certain period of time, and at the same time stabilize blood cells and prevent the release of genomic DNA, but this physical protection method has not been reduced. Sample preparation steps and added additional cost (about $6 per such special blood collection tube). Therefore, the establishment of a convenient, instant and fast cell-free nucleic acid pretreatment device is a prerequisite for reliable analysis of cell-free nucleic acid, and it can effectively deal with the huge challenges of sample processing under field conditions, which is crucial for the promotion and application of "liquid biopsy" based on cell-free nucleic acid marker detection. important.

Nucleic acid extraction and purification research based on microfluidic chips in the prior art has developed dynamic microfluidic methods including filtration, isotachophoresis, dielectrophoresis, centrifugal microfluidics, and static microfluidic methods based on immiscible phases. The concept of nucleic acid extraction and purification by immiscible phase filtration was first proposed by K.Sur and S.M.McFall et al. in 2010. This method takes advantage of the characteristics of surface tension exceeding gravitational action at the microscopic scale, and establishes between each phase interface” "Virtual Wall", which can effectively filter out impurities in one step, avoiding the steps of centrifugation and multiple washings in the traditional magnetic bead method for nucleic acid extraction; compared with the traditional magnetic bead method, this method reduces the consumption of reagents and nucleic acid. The cost of extraction simplifies the nucleic acid extraction steps, thereby shortening the nucleic acid extraction time. However, mixing is an indispensable step in nucleic acid extraction, which directly affects the efficiency and purity of extraction. Most of the existing immiscible 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.

Centrifugal microfluidic chip is one of the better technical means developed in recent years for nucleic acid POCT detection. The method of inter-chamber transfer and its use in nucleic acid extraction and purification. This method isolates the liquid in adjacent chambers through the "air gap" effect, and realizes the transfer of magnetic beads by means of external magnetic field force combined with centrifugal force. Fluidic solution for nucleic acid extraction and purification, but the maximum sample volume for processing is 200uL, which may be due to the fact that during the magnetic bead transfer process of this method, the chip is in a static state, and it may occur that the liquid in adjacent chambers is transferred through the magnetic beads under the action of gravity. The mixing of channels affects the purity of nucleic acid extraction. At the same time, free nucleic acid detection requires a large amount of original samples. Generally, whole blood samples are not less than 3 ml, and plasma samples are not less than 1.5 ml. Achieving liquid isolation between samples is somewhat difficult.

Recently Chi-Ju Kim (2018) and others designed an integrated centrifugal microfluidic chip based on solenoid valve, which can realize the integration of plasma separation and nucleic acid extraction and purification structure. Space isolation, thus realizing the extraction and purification of free nucleic acid from whole blood on a microfluidic chip. In this study, the pretreatment time of free nucleic acid was shortened to 30 minutes, but three washing steps were still included in the extraction and purification process, which may reduce the yield of nucleic acid. Rate.

SUMMARY OF THE INVENTION

In view of this, the purpose of the present invention is to provide a centrifugal microfluidic chip for free nucleic acid extraction and a method for extracting free nucleic acid. Features of high nucleic acid recovery.

The present invention provides a centrifugal microfluidic chip for free nucleic acid extraction, comprising a disc and a free nucleic acid separation and extraction processing unit located on the same side of the disc and distributed rotationally symmetrically with the center of the disc as a fixed point; the separation The extraction processing unit sequentially includes a plasma separation chamber, a nucleic acid extraction sample chamber, an immiscible phase chamber and an elution chamber; the plasma separation chamber and the nucleic acid extraction sample chamber form a loop through a passive siphon and a self-exhausting microchannel; the passive siphon One end of the passive siphon is communicated with the middle of the plasma separation chamber, and the other end of the passive siphon is communicated with one end of the nucleic acid extraction sample cavity; the middle of the passive siphon is provided with an air valve; The passive siphon extends to the center of the circle along the radial direction, and the remaining part of the passive siphon extends to the nucleic acid extraction sample cavity along the radial direction;

Between the nucleic acid extraction sample cavity and the immiscible phase cavity and/or between the immiscible phase cavity and the elution cavity, respectively, an arched capillary microchannel is used for communication; the arched capillary microchannel includes an air chamber and an air chamber. The top of the air chamber is connected to a microchannel, and the side wall of the air chamber is in sealing communication with the long arm extending from the nucleic acid extraction sample chamber, the immiscible phase chamber or the elution chamber; the length of the long arm is 3 ~6mm; the width of the bottom surface of the air chamber is 400-600 μm; the height of the connection between the side wall of the air chamber and the nucleic acid extraction sample chamber, the immiscible phase chamber or the long arm extending from the elution chamber is 150-800 μm; The microchannels extend toward the center of the circle.

Preferably, the plasma separation chamber is in the shape of a dumbbell with a thin middle and wide ends; the plasma separation chamber 1 is distributed on the disk along the radial direction; a whole blood injection hole is set at one end of the plasma separation chamber near the center of the circle ; The end of the plasma separation chamber away from the center of the circle is connected to the self-exhausting flow channel; the end of the plasma separation chamber away from the center of the circle is designed to be serrated.

Preferably, the nucleic acid extraction sample cavity is in a fan-shaped structure; the two ends of the nucleic acid extraction sample cavity are respectively provided with a sample addition hole and a sample addition hole.

Preferably, an oil filling hole is provided on the immiscible phase cavity.

Preferably, the elution chamber is provided with a hole for adding eluent.

Preferably, the volume of the plasma separation chamber is 4150 μL; the volume of the nucleic acid extraction sample chamber is 3380 μL; the volume of the immiscible phase chamber is 544 μL; the volume of the elution chamber is 153 μL.

The present invention provides a method for extracting free nucleic acid based on the centrifugal microfluidic chip, comprising the following steps:

1) adding whole blood sample, nano-magnetic bead lysis suspension, eluent and silicone oil to the plasma separation chamber, nucleic acid extraction sample chamber, elution chamber and immiscible phase chamber in sequence to obtain a sample-loading centrifugal microfluidic chip;

2) Accelerate the sample-loading centrifugal microfluidic chip to 120 rpm at an acceleration of 10 rpm/s, and then keep it for 4 min at an acceleration of 500 rpm/s until the rotational speed reaches 3600 rpm; then reduce to 350 rpm at a deceleration rate of 50 rpm/s, and then use 20 rpm/s The acceleration of s is increased to 600rpm to transfer the plasma into the nucleic acid extraction sample chamber;

3) Shake and mix the centrifugal microfluidic chip obtained in step 2) at a rotational speed of 120-840 rpm for 150 s;

4) After the centrifugal microfluidic chip is allowed to stand, place the magnet on the lower part of the centrifugal microfluidic chip, and move the sample chamber for nucleic acid extraction to the immiscible phase chamber and the elution chamber in sequence, and then move again. Shake to mix;

5) After the centrifugal microfluidic chip is allowed to stand, collect the eluate in the elution chamber.

Preferably, the addition volume of the whole blood sample is 4000 μL; the addition volume of the eluent is 50 μL and the addition volume of the silicone oil is 400 μL; the volume of the nanomagnetic bead lysis suspension is 1265 μL.

Preferably, the cell-free nucleic acid includes cfDNA, ctDNA or cell-free RNA.

The invention provides a centrifugal microfluidic chip for free nucleic acid extraction. The chip consists of two parts: one part is a plasma separation chamber suitable for plasma separation and transfer, and the other part is a nucleic acid extraction sample chamber suitable for nucleic acid separation and purification , elution chamber and immiscible phase chamber. The plasma separation chamber can be centrifugally deposited, and the use of a centrifugal microfluidic chip has the advantage of separating large volumes of plasma. The passive siphon is connected in the middle of the plasma separation chamber, which is beneficial to transfer as much plasma as possible to the nucleic acid extraction sample chamber, so as to meet the Requirements for extraction and purification of free nucleic acids.

At the same time, the key to the process of free nucleic acid extraction and purification is to ensure that all phases are not miscible during the whole process, and to ensure that a stable interface is formed during the transfer of magnetic beads. In order to achieve the above object, the present invention designs an arched capillary microchannel for magnetic bead transfer between the immiscible phases. The arched capillary microchannels form virtual water/air interfaces and air/oil interfaces according to the dominant effect of surface tension on the microscale to prevent the mixing of water and oil when the chip is stationary (during sample loading and magnetic bead transfer). ). During initiation of accelerated centrifugation, an interface of immiscible phases is formed in the microchannel. However, when the angular acceleration of the immiscible phase interface is too large, the pressure difference between the two sides of the immiscible phase interface exceeds the adaptability of the surface tension, and the interface of the immiscible phase will be destroyed, and then the water bridge will be in the adjacent Inducing fluid mixing in the chamber fails the purpose of separation and purification. In the design of the arched capillary microchannel in the present invention, the height of the formed interface is set to 200 μm, the width of the air microchannel is set to 400 μm, and under the condition that the initial acceleration is lower than 10rpm/s, the surface of the liquid and the air cavity are in contact with each other. At angles above 60°, the immiscible phase interface can always remain stable. For angular velocities above 120 rpm, the water and oil phases return to their own chambers under centrifugal force. Magnetic beads and fluid can be uniformly mixed in shaking mode in response to centrifugal and inertial forces. The superparamagnetic nanoparticles are aggregated into clusters under the action of an external magnetic field, and successively pass through several immiscible interfaces, and the magnetic force overcomes the interfacial tension to reach the elution cavity. During this process, hydrophilic and lipophilic impurities are removed, while the magnetic beads can carry the captured nucleic acid fragments into the eluent, and mixing again promotes the elution of free nucleic acid from the magnetic beads. Purified free nucleic acid fragments are obtained in the elution chamber, which can be used directly for subsequent detection or analysis. When the centrifugal microfluidic chip provided by the present invention separates free nucleic acid, compared with the method for manually extracting free nucleic acid in the prior art, the recovery rate is basically the same for whole blood samples or plasma samples, and there is no significant difference. For blood samples, the recovery rate was between 30 and 32% for high and low concentration samples, and for whole blood samples, for high and low concentration samples, the recovery rate was between 65 and 67%. It can be seen that the centrifugal microfluidic chip provided by the present invention can realize the feature of high recovery rate of free nucleic acid.

At the same time, the centrifugal microfluidic chip provided by the present invention specifically defines the heights of the water-air interface and the air-oil interface in the arched capillary microchannel, which ensures the stability of the immiscible phase interface, thereby ensuring the subsequent extraction and separation. , it can realize that a single processing unit only needs 10 minutes to process 4mL large-capacity whole blood samples each time, thus ensuring the efficient separation and extraction of samples, greatly shortening the processing time, and improving the accuracy of subsequent analysis of free nucleic acids.

Further, the centrifugal microfluidic chip provided by the present invention further restricts further optimization in the plasma separation chamber, so that the plasma separation chamber is designed to be a dumbbell-like chamber with a thin middle and wide ends, and blood cells are deposited due to the action of gravity after centrifugation. At the end away from the center of the circle, the plasma is located at the end near the center of the circle, which facilitates the plasma near the center of the circle to enter the nucleic acid extraction sample chamber through the passive siphon; an air valve is designed in the passive siphon to prevent the blood from entering under the capillary action during the sampling process. Nucleic acid extraction sample chamber.

Description of drawings

1 is a schematic diagram of a centrifugal microfluidic chip for free nucleic acid extraction provided by the present invention; wherein, 1 plasma separation chamber, 2 passive siphon, 3 self-exhausting microchannel, 4 air valve, 5 nucleic acid extraction sample chamber, 6 Immiscible phase chamber, 7 elution chambers, 8 arched capillary microchannels, 9 whole blood injection wells, 10 injection wells, 11 injection wells, 12 oil filling wells, 13 eluent wells, 14 self-venting runner;

Figure 2 is a detailed view of the arched capillary microchannel in the centrifugal microfluidic chip;

Fig. 3 is the physical diagram of the centrifugal microfluidic chip provided by the present invention;

4 is a schematic diagram and a physical diagram of different states in the process of extracting free nucleic acid using a centrifugal microfluidic chip;

5 is a graph showing the relationship between rotational speed and time in the process of extracting free nucleic acid based on a centrifugal microfluidic chip provided by the present invention;

FIG. 6 is a bar graph comparing the recovery rates of free nucleic acid extracted by the centrifugal microfluidic chip provided by the present invention and by manual extraction of free nucleic acid in the prior art.

Detailed ways

The invention provides a centrifugal microfluidic chip for free nucleic acid extraction, comprising a disc and a free nucleic acid separation and extraction processing unit located on the same side of the disc and distributed rotationally symmetrically with the center of the disc as a fixed point.

In the present invention, the disc and the free nucleic acid separation and extraction processing unit are preferably integrally formed. The number of the free nucleic acid separation and extraction processing units is not particularly limited. The number of free nucleic acid separation and extraction processing units is specifically designed according to the size of the disc, but the free nucleic acid separation and extraction processing units need to be rotationally symmetric around the center of the disc. distribution, this rotationally symmetric distribution is beneficial for the subsequent centrifugation to be free from eccentric settling. A through hole is set at the center of the disc, and the centrifugal microfluidic chip is installed on a bracket through the through hole by interference fit, and the bracket is connected with a motor to drive the centrifuge. The material of the centrifugal microfluidic chip is not particularly limited, and a material well known in the art may be used. In the embodiment of the present invention, the material of the centrifugal microfluidic chip is polymethacrylate (PMMA). In order to illustrate the design points of the centrifugal microfluidic chip, the present invention takes the example of symmetrically designing two free nucleic acid separation and extraction processing units on a disc, but this should not be understood as a limitation of the present invention.

The centrifugal microfluidic chip provided by the present invention includes a separation and extraction processing unit. The separation and extraction processing unit sequentially includes a plasma separation chamber, a nucleic acid extraction sample chamber, an immiscible phase chamber and an elution chamber. The plasma separation chamber, the nucleic acid extraction sample chamber, the immiscible phase chamber and the elution chamber are sequentially distributed on the disk near the circumference line in this order, and the sequence preferably includes counterclockwise or clockwise.

The shape of the plasma separation chamber is not particularly limited, and can be a rectangle or a trapezoid that is common in the art. In order to improve the separation rate of plasma in whole blood as much as possible, the plasma separation chamber is preferably in the shape of a dumbbell with a thin middle and wide ends; the plasma separation chamber is preferably distributed on a disc along the radial direction, and the width of the two ends is preferably Since the width of the end close to the center of the circle is smaller than the width of the end away from the center of the circle, such a design is more conducive to the deposition of blood cells in the cavity away from the center of the circle, while the upper plasma is collected in the middle part. A whole blood sampling hole is set at the end of the plasma separation chamber, preferably close to the center of the circle; the whole blood sampling hole also includes a detachable sealing cover, and the whole blood sampling hole is sealed with the sealing cover after the sampling is completed. Prevent spillage of whole blood samples. The pore size of the whole blood sample addition hole is not particularly limited, as long as it meets the requirements of sample addition. The end of the plasma separation chamber away from the center of the circle is preferably connected to the self-exhaust flow channel; the upper end of the self-exhaust flow channel faces the direction of the center of the circle, which is convenient for discharging the air in the plasma separation chamber when adding samples, and at the same time ensures the pressure of the plasma separation chamber The same as the outside atmospheric pressure. The length of the self-exhausting flow channel is preferably 0.6 to 1 times the radius of the disk, so as to prevent blood cells from overflowing from the self-exhausting flow channel during centrifugation. The pore size of the self-exhausting flow channel is not particularly limited, as long as it satisfies ventilation. The end of the plasma separation chamber away from the center of the circle is preferably designed in a zigzag shape to increase the bonding surface and ensure that the chip will not be broken during the centrifugation process. The size of the plasma separation chamber is matched with the size of the disc. Taking the diameter of the disc as 100 mm as an example, the volume of the solvent in the plasma separation chamber is preferably 4150 μL.

In the present invention, the plasma separation chamber and the nucleic acid extraction sample chamber form a circuit through a passive siphon and a self-exhausting microchannel. One end of the passive siphon tube is communicated with the middle of the plasma separation chamber, and the other end of the passive siphon tube is communicated with one end of the nucleic acid extraction sample cavity. The first 1/3 of the length of the passive siphon tube connected to the plasma separation chamber extends to the center of the circle along the radial direction, and the remaining part of the passive siphon tube extends along the radial direction to the nucleic acid extraction sample cavity. The design is beneficial to make the stratified plasma move toward the center of the circle under the inertia of centrifugal force, so as to realize the siphon effect and transfer to the nucleic acid extraction sample cavity. An air valve is arranged in the middle of the passive siphon to prevent the reagent in the sample cavity from being poured back into the plasma separation chamber. The present invention has no particular limitation on the inner diameter of the passive siphon tube, as long as it can achieve plasma transfer, and also has no particular limitation on the size of the air valve, as long as it can prevent backflow. In the embodiment of the present invention, the inner diameter of the passive siphon is 200-300 μm, and the diameter of the air valve is 1.7 mm.

In the present invention, the nucleic acid extraction sample chamber, the immiscible phase chamber and the elution chamber and their binding parts are used as a whole for the separation and purification of free nucleic acid. The function of the nucleic acid extraction sample chamber is to mix free nucleic acid in plasma with magnetic beads so that the free nucleic acid is adsorbed by the magnetic beads and removes hydrophilic impurities. So as to achieve the purpose of separating free nucleic acid from plasma. In the present invention, there is no special limitation on the shape of the nucleic acid extraction sample cavity, and a regular shape or an irregular shape common in the art can be used. In the embodiment of the present invention, in order to better utilize the space of the disc, the nucleic acid extraction sample cavity is designed to be fan-shaped. The volume of the nucleic acid extraction sample chamber is not particularly limited. When the diameter of the disc is 100 mm, the volume of the nucleic acid extraction sample chamber is 3380 μL. In order to better maintain the stability of the immiscible phase interface, a long arm is extended from the nucleic acid extraction sample cavity near the end of the immiscible phase cavity. The width of the long arm gradually narrows as it approaches the immiscible phase cavity. In the process of extending to the air cell, the width is sharply reduced to form an obtuse angle of 115° at the end near the center of the circle. The length of the long arm is preferably 3 to 6 mm, and more preferably 4.25 mm. The narrowest part of the long arm communicates with the arched capillary microchannel. The arched capillary microchannel includes an air chamber and a microchannel communicated with the top of the air chamber, and the air chamber is sealed with the narrowest part of the long arm of the nucleic acid extraction sample cavity. When magnetic beads lyse the suspension, a liquid-air interface is formed at the narrowest point (see Figure 2). The height of the liquid-air interface is 150-800 μm, more preferably 200 μm, which is beneficial to maintain the stability of the liquid-air interface. The width of the bottom surface of the air cell is 400-600 μm. The opening of the microchannel faces the center of the circle; the length of the microchannel is preferably 04 to 0.6 times the diameter of the disk. In the present invention, the inner diameter of the microchannel is not particularly limited, and the inner diameter length well known in the art can be used. The arched capillary microchannel communicates with the outside air through the microchannel. In order to improve the adaptability of the immiscible phase interface during rotation, Teflon treatment was applied to the surface of the air microchamber to reduce the surface energy and increase the interfacial tension between the air medium surface and water/oil; the method of the Teflon treatment It is preferably as follows: 0.5% Teflon AF solution is dissolved in fluorinated oil FC 77 and applied to the surface. In the examples of the present invention, the Teflon AF solution was purchased from DuPont Company of the United States, and the fluorinated oil FC 77 was purchased from 3M Company of the United States.

The immiscible phase chamber is used to add a solvent immiscible with water. The role of the immiscible phase cavity is to remove lipophilic impurities. An oil filling hole is preferably arranged on the immiscible phase cavity. The present invention has no special limitation on the shape of the immiscible phase cavity, and a common shape in the art may be used. In the embodiment of the present invention, the shape of the immiscible phase cavity is an irregular positive direction. The end of the immiscible phase cavity close to the nucleic acid extraction sample cavity preferably includes an elongated long arm, and the long arm is connected to the nucleic acid extraction sample cavity through the arched capillary microchannel to form a passage. The other end of the immiscible phase cavity preferably includes an elongated long arm, and the long arm is communicated and connected to the elution cavity through the above-mentioned arched capillary microchannel to form a passage. The characteristics of the long arm are the same as those of the long arm of the nucleic acid extraction sample cavity, which will not be repeated here. The elution chamber is used for preparing an eluent, for elution of the nucleic acid fragments adsorbed on the magnetic beads from the magnetic beads, and dissolving in the eluent. The elution chamber is provided with an eluent addition hole for adding or taking out the eluent. The diameter of the eluent addition hole has no specific requirements, as long as the eluent is added or sucked out. In the embodiment of the present invention, when the radius of the disc is 100 mm, the volume of the immiscible phase chamber is 544 μL, and the volume of the elution chamber is 153 μL.

In the present invention, the preparation method of the centrifugal microfluidic chip is preferably formed by integral casting according to the structure of the centrifugal microfluidic chip.

The present invention provides a method for extracting free nucleic acid based on the centrifugal microfluidic chip, comprising the following steps:

1) adding whole blood sample, nano-magnetic bead lysis suspension, eluent and silicone oil to the plasma separation chamber, nucleic acid extraction sample chamber, elution chamber and immiscible phase chamber in sequence to obtain a sample-loading centrifugal microfluidic chip;

2) Accelerate the sample loading centrifugal microfluidic chip with an acceleration of 10 rpm/s to 120 rpm, and then use an acceleration of 500 rpm/s until the rotation speed reaches 3600 rpm for 4 min; then reduce to 350 rpm with a deceleration of 50 rpm/s, and then use 20 rpm/s. The acceleration is increased to 600rpm to transfer the plasma into the nucleic acid extraction sample chamber;

3) Shake and mix the centrifugal microfluidic chip obtained in step 2) at a rotational speed of 120-840 rpm for 150 s;

4) After the centrifugal microfluidic chip is allowed to stand, place the magnet on the lower part of the centrifugal microfluidic chip, and move the sample chamber for nucleic acid extraction to the immiscible phase chamber and the elution chamber in sequence, and then move again. Shake to mix;

5) After the centrifugal microfluidic chip is allowed to stand, collect the eluate in the elution chamber.

In the present invention, whole blood sample, nano-magnetic bead lysis suspension, eluent and silicone oil are sequentially added to the plasma separation chamber, nucleic acid extraction sample chamber, elution chamber and immiscible phase chamber to obtain a sample adding centrifugal microfluidic chip.

The source of the whole blood sample is not particularly limited in the present invention, and a well-known whole blood sample in the art may be used. The whole blood sample preferably includes cfDNA, ctDNA or cell-free RNA. The nanomagnetic bead lysis suspension includes a nanomagnetic bead magnetic bead suspension and a lysis/binding buffer. The nano-magnetic beads are silica-coated nano-magnetic beads. In the embodiment of the present invention, the source of the silica-coated nano-magnetic beads is a kit, which is purchased from Thermo Fisher; MagMAX Cell-Free Total Nucleic Acid Kit (A36716, ThermoFisher, USA ). The source of the lysis/binding buffer was a kit, which was purchased from Thermo Fisher; MagMAX Cell-Free TotalNucleic Acid Kit (A36716, ThermoFisher, USA). Taking the specifications of the centrifugal microfluidic chip prepared above as an example, the addition volume of the whole blood sample is preferably 4000 μL; the addition volume of the eluent is preferably 50 μL and the addition volume of the silicone oil is preferably 400 μL; the nano-magnetic bead lysis suspension The volume is preferably 1265 μL.

After adding the sample, the present invention keeps the sample-loading centrifugal microfluidic chip at an acceleration of 10rpm/s until the rotational speed reaches 3600rpm for 4min; then decreases to 350rpm at a deceleration of 50rpm/s, and then increases to 600rpm at an acceleration of 20rpm/s. The plasma is transferred to the nucleic acid extraction sample chamber.

In the present invention, preferably, the sample-loading centrifugal microfluidic chip is mounted on the support, the engine is turned on, and centrifugation is performed. The rotation direction at an acceleration of 500rpm/s until the rotation speed reaches 3600rpm is limited to rotation according to the distribution direction of the plasma separation chamber, the nucleic acid extraction sample chamber, the immiscible phase chamber and the elution chamber, otherwise the purpose of separation and extraction cannot be achieved. The rotation speed of 3600rpm can realize the solid-liquid separation of whole blood. The plasma was transferred to the nucleic acid extraction sample chamber at a deceleration rate of 50rpm/s to 350rpm and then increased to 600rpm at an acceleration of 20rpm/s. The control of the acceleration and deceleration is beneficial to pass the passive siphon in the chip to make the separated plasma in the inertial effect. down into the nucleic acid extraction sample cavity to achieve sufficient transfer of plasma.

After the separated plasma is transferred to the nucleic acid extraction sample chamber, the present invention shakes and mixes the centrifugal microfluidic chip at a speed of 120-840 rpm for 150 s.

In the present invention, the method of shaking and mixing is preferably as follows: when shaking, the rotating speed is in the same direction, but the shaking process is acceleration and deceleration back and forth. The purpose of the shaking and mixing is to promote the sufficient reaction and combination of the nanomagnetic beads and the nucleic acid to form a magnetic bead-nucleic acid complex.

After the centrifugal microfluidic chip is allowed to stand, the present invention places the magnet on the lower part of the centrifugal microfluidic chip, and moves from the position of the nucleic acid extraction sample chamber to the immiscible phase chamber and the elution chamber in sequence, and then again. Shake to mix.

In the present invention, the magnet provides a magnetic force so as to attract the magnetic beads. The moving speed of the magnet is preferably 1 to 5 mm/s, more preferably 2 to 4 mm/s, and most preferably 3 mm/s. The method of moving the magnet includes manual movement or mechanical movement.

In the present invention, the method of shaking and mixing again is the same as the method of shaking and mixing above, and will not be repeated here. The purpose of shaking and mixing again is that the nucleic acid-adsorbing magnetic beads entering the elution chamber will elute the nucleic acid in the eluent. Shaking is beneficial to speed up the elution process, so that the adsorbed nucleic acid can be eluted as much as possible and dissolved until it is washed. in dehydration.

After elution, the present invention collects the eluent in the elution chamber after the centrifugal microfluidic chip is allowed to stand.

In the present invention, the method for collecting the eluate in the elution chamber preferably adopts a micropipette to suck.

In the present invention, the collected eluate is enriched with cfDNA or ctDNA in whole blood, and subsequent detection of the enriched cfDNA or ctDNA is carried out by means of analysis. It can be seen that the method provided by the present invention simplifies the steps of extracting free nucleic acid from whole blood At the same time, the processing time is shortened to ensure that the free nucleic acid in the collected whole blood will not be degraded, providing a basis for medical testing.

A centrifugal microfluidic chip for free nucleic acid extraction provided by the present invention and a method for extracting free nucleic acid provided by the present invention will be described in detail below with reference to the examples, but they should not be construed as limiting the protection scope of the present invention.

Example 1

A method for extracting a centrifugal microfluidic chip for free nucleic acid extraction, comprising the following steps:

(1) The centrifugal microfluidic chip is mounted on the bracket, and the bracket is electrically connected to the motor. Four groups of samples (including low-concentration whole blood, high-concentration whole blood, low-concentration plasma, and high-concentration plasma samples were used, in which the low concentration in two samples was 10 ¹ copies/ml; the high concentration in two samples was 10 ⁵ copies/ml. ) were added to the plasma separation chamber, respectively, and 15 μL of the silica-coated nanomagnetic bead magnetic bead suspension and 1.25 mL of lysis/binding buffer were added to the nucleic acid extraction sample chamber through the loading hole on the nucleic acid extraction sample chamber. Add 50 μL of eluate to the elution chamber. Add 400 μL of silicone oil to the immiscible phase chamber.

(2) Start the motor power supply, accelerate to 120 rpm at an acceleration of 10 rpm/s, and then continue at an acceleration of 500 rpm/s until 3600 rpm for 4 min, and separate the plasma from the cells. Then, decelerating at 50 rpm/s to 350 rpm to activate the siphon valve, and then at an acceleration of 20 rpm/s until the rotational speed reached 600 rpm, about 1.6 mL of plasma was transferred to the sample chamber by inertia for cfDNA extraction.

(3) The chip is shaken and mixed for 150s between 120rpm and 840rpm to promote the nano-magnetic beads and nucleic acid to fully react and combine to form a magnetic bead-nucleic acid complex. The relationship between the time and rotation speed of the shaking and mixing mode is shown in Figure 5.

(4) Place the hand-held magnet on the lower part of the chip, move from the nucleic acid extraction sample chamber to the immiscible phase chamber at a speed of 3 mm/s, and then move from the immiscible phase chamber to the elution chamber, so that the magnetic bead-nucleic acid complex is Elution is carried out through the arched capillary microchannel into the elution chamber.

(5) Implement the shaking and mixing mode between 120rpm and 840rpm again for 150s to promote the sufficient elution of nucleic acids from the magnetic nanobeads.

(6) The magnetic beads are adsorbed on the bottom of the elution chamber by the magnet, the supernatant of the elution buffer is collected by a micropipette, and the recovery rate of free nucleic acid is counted (quantitative detection of the recovered samples, the ratio of the two is Recovery rate).

At the same time, in order to verify the effect of the method provided by the present invention on the extraction of free nucleic acid, the applicant used manual method ((https://www.thermofisher.com/order/catalog/product/A36716) to extract the free nucleic acid of the same sample, using After the recovery was determined in the same way, a recovery histogram was drawn.

The results are shown in Figure 6. It can be seen from Fig. 6 that when the centrifugal microfluidic chip is used for free nucleic acid separation, compared with the method of manually extracting free nucleic acid in the prior art, the recovery rates for both whole blood samples and plasma samples are basically the same, and there is no significant difference. For whole blood samples, the recoveries were between 30% and 32% for high concentration samples and low concentration samples, and between 65% and 67% for whole blood samples for high concentration samples and low concentration samples.

The above are only the preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, without departing from the principles of the present invention, several improvements and modifications can be made. It should be regarded as the protection scope of the present invention.

Claims (9)

1. A centrifugal microfluidic chip for extracting free nucleic acid is characterized by comprising a disc and separation and extraction processing units which are positioned on the disc and are rotationally and symmetrically distributed;

the separation extraction processing unit comprises a plasma separation chamber, a nucleic acid extraction sample cavity, an immiscible phase cavity and an elution cavity in sequence; the plasma separation chamber and the nucleic acid extraction sample cavity form a loop through a passive siphon and a self-venting micro-channel; one end of the passive siphon is communicated with the middle part of the plasma separation chamber, and the other end of the passive siphon is communicated with the nucleic acid extraction sample cavity; an air valve is arranged in the middle of the passive siphon; a passive siphon tube which is 1/3 long before one end communicated with the plasma separation chamber extends to the circle center along the radius direction, and the rest part of the passive siphon tube extends to the nucleic acid extraction sample cavity along the radius direction;

the nucleic acid extraction sample cavity and the immiscible phase cavity and/or the immiscible phase cavity and the elution cavity are communicated by adopting an arch capillary micro-channel respectively; the arch-shaped capillary microchannel comprises an air chamber and a microchannel communicated with the top of the air chamber, wherein the side wall of the air chamber is in sealed communication with a long arm extending from a nucleic acid extraction sample cavity, an immiscible phase cavity or an elution cavity; the length of the long arm is 3-6 mm; the width of the bottom surface of the air cell is 400-600 mu m; the height of the joint of the side wall of the air cell and the extended long arm of the nucleic acid extraction sample cavity, the immiscible phase cavity or the elution cavity is 150-800 μm; the micro-channel extends towards the direction of the circle center;

the long arm extends out of the nucleic acid extraction sample cavity near the end of the immiscible phase cavity, the width of the long arm gradually narrows along the direction of the immiscible phase cavity, the width sharply decreases in the process of extending to the small air chamber, an obtuse angle of 115 degrees is formed at the end near the center of the circle, and the characteristics of the long arm at the two ends of the immiscible phase cavity are the same as those of the long arm of the nucleic acid extraction sample cavity.

2. The centrifugal microfluidic chip according to claim 1, wherein the plasma separation chamber is dumbbell-like with a thin middle part and two wide ends; the plasma separation chambers are distributed on the disc along the radius direction; a whole blood sample adding hole is formed in one end, close to the circle center, of the plasma separation chamber; one end of the plasma separation chamber, which is far away from the circle center, is communicated with a self-exhaust flow passage; one end of the plasma separation chamber far away from the circle center is designed to be serrated.

3. The centrifugal microfluidic chip of claim 1, wherein the nucleic acid extraction sample chamber is a fan-shaped structure; two ends of the nucleic acid extraction sample cavity are respectively provided with a sample adding hole.

4. The centrifugal microfluidic chip according to claim 1, wherein the immiscible phase chamber is provided with an oil filling hole.

5. The centrifugal microfluidic chip according to claim 1, wherein the elution chamber is provided with an elution solution adding hole.

6. The centrifugal microfluidic chip of any one of claims 1 to 5, wherein the volume of the plasma separation chamber is 4150 μ L; the volume of the nucleic acid extraction sample cavity is 3380 mu L; the volume of the immiscible phase cavity is 544 μ Ι _; the volume of the elution chamber was 153. mu.L.

7. A method for extracting free nucleic acid based on the centrifugal microfluidic chip of any one of claims 1 to 6 for non-diagnostic purposes, comprising the steps of:

1) sequentially adding a whole blood sample, a nano magnetic bead cracking suspension, an eluent and silicone oil to the plasma separation chamber, the nucleic acid extraction sample cavity, the elution cavity and the immiscible phase cavity to obtain a sample-adding centrifugal microfluidic chip;

2) accelerating the sample-adding centrifugal microfluidic chip to 120rpm at the acceleration of 10rpm/s, and then maintaining the chip for 4min at the acceleration of 500rpm/s until the rotating speed reaches 3600 rpm; then the speed is reduced to 350rpm at the deceleration of 50rpm/s, and then the speed is increased to 600rpm at the acceleration of 20rpm/s so that the plasma is transferred to the nucleic acid extraction sample cavity;

3) shaking and uniformly mixing the centrifugal micro-fluidic chip obtained in the step 2) for 150s at the rotating speed of 120-840 rpm;

4) after the centrifugal microfluidic chip is stood, placing a magnet at the lower part of the centrifugal microfluidic chip, moving the position of the nucleic acid extraction sample cavity to the direction of the immiscible phase cavity and the elution cavity in sequence, and shaking and mixing uniformly again;

5) and after the centrifugal microfluidic chip is stood, collecting the eluent in the elution cavity.

8. The method of claim 7, wherein the added volume of the whole blood sample is 4000 μ L; the addition volume of the eluent is 50 mu L and the addition volume of the silicone oil is 400 mu L; the volume of the nano magnetic bead lysis suspension is 1265. mu.L.

9. The method of claim 7 or 8, wherein the episomal nucleic acid comprises ctDNA, cfDNA, or episomal RNA.

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