Integrated multi-target detection micro-fluidic chip

Abstract

The integrated multi-target detection micro-fluidic chip comprises an upper layer and a lower layer, the upper layer is a chip layer (1), and the lower layer is a rotary magnetic control layer (8); the chip layer (1) is composed of a nucleic acid extraction chamber (3) located in the center, and a plurality of first washing chambers (4), second washing chambers (5) and elution / multi-target amplification detection chambers (6) which are arranged around the circumference of the nucleic acid extraction chamber (3) at equal intervals and are communicated with one another; the rotary magnetic control layer (8) is composed of a pluggable rotary disc (10) and magnetic bars (9) arranged on the rotary disc (10) in a concentric circle mode. According to the present invention, the structure design is simple, the integrated function of sample lysis, nucleic acid purification and multi-target nucleic acid simultaneous detection can be achieved without the complex valve device, the time and the labor are saved, the multi-sample and multi-target high-throughput rapid detection can be easily achieved, and the good application prospect is provided.

Description

Technical Field

[0001] The present invention belongs to the field of nucleic acid detection, and in particular relates to an integrated multi-target detection microfluidic chip. Background Art

[0002] In traditional nucleic acid detection technology, multiple steps are usually required to complete the detection of pathogens, including sample lysis, nucleic acid extraction and purification, nucleic acid amplification and detection. These steps usually need to be completed in different equipment, resulting in complex processes, time-consuming operations and high costs. Traditional nucleic acid detection methods can often only detect a single pathogen target and cannot meet the needs of simultaneous detection of multiple targets.

[0003] In recent years, polymerase chain reaction (PCR) technology has been widely used in the biomedical field as an efficient and sensitive nucleic acid amplification method. PCR technology can replicate DNA fragments in vitro, thereby greatly increasing the amount of target DNA and making it easier to detect the target DNA. In addition, PCR technology is highly specific and can accurately detect target sequences in complex samples, making PCR technology one of the important tools for pathogen detection.

[0004] However, the existing PCR technology can usually only detect a single pathogen and cannot meet the needs of simultaneous detection of multiple pathogens in practical applications. At the same time, the sample extraction step is also an indispensable part of the nucleic acid detection process. The traditional manual extraction operation is not only time-consuming and labor-intensive, but also has the risk of sample cross-contamination. Magnetic bead-based nucleic acid extraction is generally divided into four steps: lysis, binding, washing, and elution. Compared with traditional nucleic acid purification methods, the extraction process is simple, efficient, safe and non-toxic, and can be automated. It is suitable for high-throughput, automated extraction of large quantities of clinical samples. However, the existing method of manually dragging or automatically operating a magnet to drag the magnetic beads to move and mix in the chamber causes the magnetic beads to move in groups, which may cause insufficient mixing and unsatisfactory capture of nucleic acids by the magnetic beads. Therefore, it is necessary to develop a device that completes sample pretreatment, PCR amplification, and detection of multiple pathogen targets on the same platform to meet the needs of practical applications. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide an integrated multi-target detection microfluidic chip to overcome the defects of the prior art nucleic acid extraction and purification device, such as complicated detection operation, long detection time, high degree of manualization, low extraction efficiency and single detection target.

[0006] The present invention provides an integrated multi-target detection microfluidic chip, comprising an upper layer and a lower layer, wherein the upper layer is a chip layer and the lower layer is a rotating magnetic control layer; the chip layer is composed of a nucleic acid extraction chamber located in the center and a plurality of interconnected first washing chambers, second washing chambers, and elution / multi-target amplification detection chambers equally arranged around the circumference of the nucleic acid extraction chamber; the rotating magnetic control layer is composed of an insertable turntable and a plurality of magnetic rods concentrically arranged on the turntable.

[0007] The nucleic acid extraction chamber is located in the center of the chip layer. Compared with other chambers, it has a larger volume and can hold large-volume samples and corresponding proportions of lysate. The first washing chamber, the second washing chamber, and the elution / multi-target amplification detection chamber can be understood as a unit, and multiple units are equally divided around the circumference of the nucleic acid extraction chamber; the unit composed of the first washing chamber, the second washing chamber, and the elution / multi-target amplification detection chamber can react synchronously after being equally divided in the nucleic acid extraction chamber.

[0008] Preferably, a glass substrate is provided between the chip layer and the rotating magnetron layer.

[0009] Preferably, the first washing chamber is connected to the nucleic acid extraction chamber through a micro-column array to form a stable water-oil interface.

[0010] Preferably, a motor shaft socket is provided in the center of the turntable.

[0011] Preferably, the arrangement of the plurality of magnetic bars on the turntable is divided into an inner layer and an outer layer; the inner layer magnetic bars are equally spaced around the center of the turntable; the outer layer magnetic bars are arranged correspondingly according to the arrangement positions of the first washing chamber, the second washing chamber, and the elution / multi-target amplification detection chamber. The outer layer magnetic bars are used for the magnetic beads to change their diameter and move around the circle to pass through the first washing chamber, the second washing chamber, and the elution / multi-target amplification detection chamber in turn. A certain gap is left between the upper chip layer and the lower rotating magnetic control layer to ensure that the lower layer rotates and the upper layer remains fixed, and the magnetic bars still have a strong control over the magnetic beads.

[0012] Preferably, the inner layer magnetic bars and the outer layer magnetic bars are arranged alternately and staggered to minimize the mutual influence between the outer circle magnetic field and the inner circle magnetic field.

[0013] Preferably, the nucleic acid extraction chamber is filled with a magnetic bead solution and a nucleic acid extraction reagent. Preferably, the first washing chamber and the second washing chamber are filled with a washing solution; the elution / multi-target amplification detection chamber is filled with an amplification reagent and an elution solution. Except for the nucleic acid extraction chamber, the other chamber channels are filled with mineral oil, and the aqueous reagent in each reaction chamber is used as a water phase to form droplets in the oil phase.

[0014] Preferably, the microfluidic chip is fixed by building a platform bracket when in use.

[0015] Preferably, the microfluidic chip controls the rotation of the turntable by a stepping motor when in use.

[0016] Preferably, the channel width between the nucleic acid extraction chamber and the first washing chamber and the gap width between the microcolumns are appropriately increased to ensure that the surface tension prevents the solution in the nucleic acid extraction chamber from entering the first washing chamber, the second washing chamber, and the elution / multi-target amplification detection chamber, while allowing the magnetic beads to pass through the channel into the first washing chamber with less hindrance.

[0017] The present invention is based on the magnetic bead method nucleic acid extraction technology, and is mainly divided into two layers, the upper layer is the chip layer, and the lower layer is the rotating magnetic control layer. The magnets are arranged in a circle and designed to be a turntable, and the movement of the magnetic beads is manipulated by the rotation of the motor to realize specific functions in each chamber of the upper layer, so that the degree of automation is greatly improved. More importantly, under the action of the permanent magnet, the rotating magnetic control layer drives the fluid to move, generates vortices, and forms an active mixer, which can further improve the efficiency of magnetic beads in capturing nucleic acids and make their lysis more complete. Finally, the purpose of magnetic beads being evenly divided is achieved when stationary, and it is capable of detecting multiple pathogens at the same time, and achieving the expected effect of single sample multi-target detection. Compared with traditional manual operation, this automated extraction method not only greatly improves the efficiency of nucleic acid capture, but also effectively avoids the risk of sample cross-contamination, thereby ensuring the accuracy and reliability of the test results.

[0018] The present invention integrates nucleic acid extraction, purification and amplification into a chip. The magnetic beads are fully mixed in the lysate under the action of a rotating magnetic control device, and the nucleic acids are evenly divided by standing. Then, they are attracted by the outer ring magnet on the turntable, enter the washing chamber, continue to move in a circle, and finally reach the amplification chamber to perform an independent amplification reaction, thereby realizing the complete process of nucleic acid extraction, purification and amplification, thereby realizing the detection of multiple pathogen targets in a single sample. During operation, the code can be edited in advance to control the speed and time of the motor rotation. The entire nucleic acid extraction process does not require human intervention. The motor rotation drives the lower disk to rotate and drag the magnetic beads, so that the magnetic beads shuttle through various reaction reagents to complete the detection.

[0019] Beneficial Effects

[0020] (1) The present invention uses the principle of magnetic field driven magnetic bead transfer to extract and purify nucleic acids through the unique design of the chip. The added lower rotating magnetic control layer, under the action of the permanent magnet, the magnetic beads rotate to drive the fluid movement, generate vortices, and form an active mixer. Due to the intense and full contact between the magnetic beads and the solution, the magnetic bead capture efficiency is greatly improved. After the code is designed, the entire process does not require manual intervention, and the subsequent combination of the heating module makes the present invention fully automated. After rotating and standing, the magnetic beads can automatically form an even distribution, and then under the action of the magnetic rod, the magnetic beads are simultaneously dragged into an independent washing and amplification chamber to achieve simultaneous detection of multiple targets in a single sample. The chip structure design is simple, and no complex valve device is required. It can realize the functions of sample lysis, nucleic acid purification and simultaneous detection of multiple target nucleic acids in one, saving time and effort, and more easily realizing high-throughput rapid detection of multiple samples and multiple targets. (2) The present invention has strong universality and can detect multiple pathogens at the same time, saving time and effort. It can play a good screening role in the initial detection process. It only needs to be driven by a motor to complete nucleic acid extraction, even distribution, purification and elution, and then it can be directly transferred to a flat-plate PCR instrument for amplification and detection steps.

[0021] (3) The present invention adopts a method of high-speed rotation and back and forth swinging and two washings, so that the magnetic bead group carries very few interfering substances, greatly improving the extraction and purification effect of nucleic acid, making the detection more sensitive and accurate. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is a plan view of the chip layer of the integrated multi-target detection microfluidic chip of the present invention.

[0023] Figure 2 It is a stereoscopic diagram of the chip layer of the integrated multi-target detection microfluidic chip of the present invention.

[0024] Figure 3 It is a plan view of the rotating magnetic control layer of the integrated multi-target detection microfluidic chip of the present invention.

[0025] Figure 4 This is a stereoscopic diagram of the rotating magnetic control layer of the integrated multi-target detection microfluidic chip of the present invention.

[0026] Figure 5 This is a schematic diagram of the rotating magnetic control layer of the integrated multi-target detection microfluidic chip of the present invention being installed on the motor of the building platform.

[0027] Figure 6 This is a plan view of the combination of the chip layer and the rotating magnetic control layer of the integrated multi-target detection microfluidic chip of the present invention.

[0028] Figure 7 This is a three-dimensional diagram of the combination of the chip layer and the rotating magnetic control layer of the integrated multi-target detection microfluidic chip of the present invention.

[0029] Figure 8 This is a top view of the integrated multi-target detection microfluidic chip of the present invention installed on a building platform.

[0030] Fig. 9 This is a side view of the integrated multi-target detection microfluidic chip of the present invention installed on a building platform.

[0031] Fig.10 This is a three-dimensional diagram of the integrated multi-target detection microfluidic chip of the present invention installed on a building platform.

[0032] Fig.11 This is a graph showing the results of pathogen nucleic acid detection using the integrated multi-target detection microfluidic chip of the present invention. DETAILED DESCRIPTION

[0033] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0034] Example 1

[0035] This embodiment provides an integrated multi-target detection microfluidic chip, including an upper layer and a lower layer:

[0036] like Figure 1 and Figure 2 As shown, the upper layer is the chip layer 1, and the lower layer is the rotating magnetron layer 8; the chip layer 1 is composed of a nucleic acid extraction chamber 3 located in the center and a plurality of interconnected first washing chambers 4, second washing chambers 5, and elution / multi-target amplification detection chambers 6 equally arranged around the circumference of the nucleic acid extraction chamber 3. The first washing chamber 4 is connected to the nucleic acid extraction chamber 3 through a micro-column array 7. Preferably, the nucleic acid extraction chamber 3 is filled with a magnetic bead solution and a nucleic acid extraction reagent. The first washing chamber 4 and the second washing chamber 5 are filled with a washing solution; the elution / multi-target amplification detection chamber 6 is filled with an amplification reagent and an elution solution.

[0037] like Figure 3 and Figure 4As shown, the rotating magnetic control layer 8 is composed of an insertable turntable 10 and a plurality of magnetic rods 9 arranged in concentric circles on the turntable 10. A motor shaft plug hole 12 is provided in the center of the turntable 10. The plurality of magnetic rods 9 correspond to a plurality of magnetic rod plug holes 11. The arrangement of the plurality of magnetic rods 9 on the turntable 10 is divided into an inner layer and an outer layer; the inner layer magnetic rods 91 are arranged in equal parts around the center of the turntable 10; the outer layer magnetic rods 92 are arranged correspondingly according to the arrangement positions of the first washing chamber 4, the second washing chamber 5, and the elution / multi-target amplification detection chamber 6. Preferably, the inner layer magnetic rods 91 and the outer layer magnetic rods 92 are arranged alternately.

[0038] like Figure 7 As shown, a glass substrate 2 is provided between the chip layer 1 and the rotating magnetron layer 8 .

[0039] like Figure 5-6 As shown in 8-10, the microfluidic chip is fixed by building a platform bracket 13 when in use. The turntable 10 is controlled to rotate by a stepping motor 14 when the microfluidic chip is in use.

[0040] This embodiment also provides a specific method for using an integrated multi-target detection microfluidic chip, comprising the following steps: 1) adding nucleic acid extraction reagents, magnetic beads, and samples to be detected into the nucleic acid extraction chamber 3 .

[0041] 2) Mineral oil is filled between the channels of the first washing chamber 4, the second washing chamber 5, and the elution / multi-target amplification detection chamber 6, and then different washing reagents are added to the first washing chamber 4 and the second washing chamber 5 respectively; different PCR amplification reagents and elution solutions are added to the elution / multi-target amplification detection chamber 6 to form oil-in-water droplets.

[0042] 3) Connect the motor to drive the lower turntable 10 to move, so that the magnetic beads in the nucleic acid extraction chamber 3 rotate at high speed to drive the fluid to move, generate a vortex, form an active mixer, promote the sample lysis reaction and enrich more nucleic acids on each magnetic bead. Then stop the motor and let it stand.

[0043] 4) The magnetic beads carrying nucleic acid are evenly divided under the action of the inner magnetic rod 91, and the motor rotates again to a certain angle and stops, so that the outer magnetic rod 92 is transferred to the first washing chamber 4, and the magnetic beads are slowly sucked into the first washing chamber 4 by magnetic force.

[0044] 5) After each portion of magnetic beads completely enters the first washing chamber 4, the motor drives the magnetic disk to swing back and forth quickly in a small range to fully wash the magnetic beads to remove impurities (the impurities include impurities other than nucleic acids such as proteins and fragments enriched on the magnetic beads after sample lysis, which will affect the nucleic acid detection results and therefore need to be removed);

[0045] 6) The turntable 10 rotates, and the magnetic bar 9 drags the magnetic beads in the first washing chamber 4 into the second washing chamber 5 respectively, completing the second washing, and the back and forth swinging process is the same as above;

[0046] 7) The turntable 10 rotates, dragging the magnetic beads in the second washing chamber 5 into the elution / multi-target amplification detection chamber 6 respectively. The turntable 10 swings slightly and quickly as above, so that the magnetic beads and the elution solution are fully in contact and vibrated, so that the nucleic acid is eluted. The turntable 10 rotates in the opposite direction to drag the magnetic beads back to the second washing chamber 5; the chip is subjected to amplification reaction and then observed under a fluorescence microscope.

[0047] Fig.11 The results of nucleic acid detection of pathogens by the integrated multi-target detection microfluidic chip of the present invention include human parainfluenza virus (FAM), influenza A (HEX) and rhinovirus (ROX). Fig.11 It can be seen that according to the fluorescence intensity difference measurement after amplification (the fluorescence intensity of the positive well is higher than that of the blank control), the pathogen lysis, nucleic acid purification and amplification of this system can realize the nucleic acid detection of various pathogens such as human parainfluenza virus, influenza A and rhinovirus.

Claims

1. An integrated multi-target detection microfluidic chip, comprising an upper layer and a lower layer, characterized in that: The upper layer is a chip layer (1), and the lower layer is a rotating magnetron layer (8); the chip layer (1) is composed of a nucleic acid extraction chamber (3) located in the center and a plurality of interconnected first washing chambers (4), second washing chambers (5), and elution / multi-target amplification detection chambers (6) arranged equally around the circumference of the nucleic acid extraction chamber (3); the rotating magnetron layer (8) is composed of an insertable turntable (10) and a plurality of magnetic rods (9) arranged in concentric circles on the turntable (10).

2. The integrated multi-target detection microfluidic chip according to claim 1, characterized in that: A glass substrate (2) is provided between the chip layer (1) and the rotating magnetron layer (8).

3. The integrated multi-target detection microfluidic chip according to claim 1, characterized in that: The first washing chamber (4) is connected to the nucleic acid extraction chamber (3) via a micro-column array (7).

4. The integrated multi-target detection microfluidic chip according to claim 1, characterized in that: A motor shaft insertion hole (12) is provided in the center of the rotating disk (10).

5. The integrated multi-target detection microfluidic chip according to claim 1, characterized in that: The arrangement of the plurality of magnetic bars (9) on the turntable (10) is divided into an inner layer and an outer layer; the inner layer magnetic bars (91) are arranged in equal parts around the center of the turntable (10); and the outer layer magnetic bars (92) are arranged corresponding to the arrangement positions of the first washing chamber (4), the second washing chamber (5), and the elution / multi-target amplification detection chamber (6).

6. The integrated multi-target detection microfluidic chip according to claim 5, characterized in that: The inner layer magnetic bars (91) and the outer layer magnetic bars (92) are arranged alternately and staggered.

7. The integrated multi-target detection microfluidic chip according to claim 1, characterized in that: The nucleic acid extraction chamber (3) is filled with a magnetic bead solution and a nucleic acid extraction reagent.

8. The integrated multi-target detection microfluidic chip according to claim 1, characterized in that: The first washing chamber (4) and the second washing chamber (5) are filled with washing solutions; and the elution / multi-target amplification detection chamber (6) is filled with amplification reagents and elution solutions.

9. The integrated multi-target detection microfluidic chip according to claim 1, characterized in that: The microfluidic chip is fixed by building a platform support (13) when in use.

10. The integrated multi-target detection microfluidic chip according to claim 1, characterized in that: When the microfluidic chip is in use, the rotation of the turntable (10) is controlled by a stepping motor (14).

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