Automatic electromagnetic micro-fluidic chip for nucleic acid purification and use method of automatic electromagnetic micro-fluidic chip

Abstract

The invention relates to an automatic electromagnetic micro-fluidic chip for nucleic acid purification and a use method of the automatic electromagnetic micro-fluidic chip. The automatic electromagnetic micro-fluidic chip comprises a multi-channel micro-fluidic detection chip and an automatic magnet control system positioned below the chip, the automatic magnet control system comprises a PCB electromagnetic coil and a plurality of permanent magnets placed above the PCB electromagnetic coil. And the coil units of the PCB electromagnetic coil are in one-to-one correspondence with the chamber units of the microfluidic detection chip in position. According to the micro-fluidic chip, automation, integration and miniaturization of nucleic acid samples from pretreatment to extraction and purification can be achieved, meanwhile, the advantages of the micro-fluidic chip are fused, and the complex nucleic acid extraction process can be integrated into the small chip through the accurately designed micro-channel and the micro-cavity. The whole nucleic acid extraction process can be completed by a user only through simple sample loading and some basic operations, and the method has a good market application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of microfluidic chips, and in particular relates to an automated electromagnetic microfluidic chip for nucleic acid purification and a use method thereof. Background Art

[0002] Nucleic acid extraction and purification is a prerequisite for many genetic tests and genetic diagnoses. High-quality nucleic acid samples can ensure the accuracy and sensitivity of subsequent tests. Currently, nucleic acid extraction and purification methods can be divided into two categories: filter membrane-based methods and magnetic bead-based methods.

[0003] Nucleic acid extraction based on filter membranes uses the interaction between nucleic acids and filter membranes to achieve adsorption and elution, thereby separating and purifying nucleic acids. This method is easy to operate, but the filtration efficiency is poor, the possibility of reuse is not high, and it is usually difficult to obtain high-purity nucleic acid products. In addition, the problem of filter membrane clogging also seriously restricts its automated application. In contrast, the nucleic acid extraction technology based on magnetic beads has higher specificity and stronger binding force. It has a good adsorption effect on nucleic acids under certain conditions (such as under a chaotropic salt system), and can quickly separate and release nucleic acids when conditions change. It is a good tool for nucleic acid enrichment, extraction, and purification. Currently, there are many types of commercial magnetic bead products with different functions, which can specifically capture different types of nucleic acids such as DNA and RNA. However, this method also requires a lot of manual washing, centrifugation and other complicated operations, and the degree of automation is limited.

[0004] Microfluidic chip technology provides the possibility of realizing a new method of automated nucleic acid extraction. Microfluidic chips use micron-scale channels to precisely control fluids, which can highly integrate many biochemical functions and realize the automation of complex operations. Currently, microfluidic chips have been widely used in protein separation and detection, pathogen diagnosis, and drug screening. However, there are few reports on the use of microfluidic chips to fully automate the process from sample pretreatment to product purification. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide an automated electromagnetic microfluidic chip for nucleic acid purification and a method of using the same, wherein the microfluidic chip can realize automation, integration and miniaturization of nucleic acid sample pretreatment to extraction and purification.

[0006] The present invention provides an automated electromagnetic microfluidic chip for nucleic acid purification, comprising a multi-channel microfluidic detection chip and an automated magnet control system located below the chip; the automated magnet control system comprises a PCB electromagnetic coil and a plurality of permanent magnets placed above the PCB electromagnetic coil; the coil units of the PCB electromagnetic coil correspond one-to-one to the positions of the chamber units of the multi-channel microfluidic detection chip.

[0007] Preferably, the chamber unit of the multi-channel microfluidic detection chip includes a lysis liquid chamber, a first washing liquid chamber, a second washing liquid chamber and an elution liquid chamber, that is, a single channel.

[0008] Preferably, the multi-channel microfluidic detection chip is prepared by pouring PDMS into an aluminum alloy mold, attaching a plastic film to the surface and then heating and curing it, and after molding, using a plasma bonding device to clean and bond the PDMS base and the glass substrate to obtain a multi-channel microfluidic detection chip.

[0009] Preferably, the PCB electromagnetic coil adopts FR-4 insulating substrate material and is a planar solenoid structure formed by stacking multiple layers.

[0010] Preferably, the material of the permanent magnet is N52.

[0011] The present invention also provides a method for using an automated electromagnetic microfluidic chip for nucleic acid purification, comprising the following steps:

[0012] (1) firstly, pre-filling the aqueous elution reagent into the elution liquid chamber, then injecting mineral oil into the lysis liquid chamber, tilting the chip multi-channel microfluidic detection chip to allow the mineral oil to flow through the bottom pipeline until the first washing liquid chamber and the second washing liquid chamber are filled; finally, using a pipette gun to inject the lysis liquid, the first washing liquid, and the second washing liquid into the corresponding chambers;

[0013] (2) adding the sample to the lysate chamber, driving the magnetic beads to mix the reagent by a permanent magnet and letting it stand, waiting for the magnetic beads to fully adsorb the lysed nucleic acid; then driving the magnetic beads through the channel at the bottom to enter the first washing liquid chamber, and similarly enter the second washing liquid chamber, and attracting the magnetic beads to vibrate back and forth by the magnet; then attracting the magnetic beads to the elution liquid chamber, mixing and letting it stand, so that the nucleic acid is fully eluted in the aqueous elution reagent; finally, attracting the magnetic beads back to the second washing liquid chamber, completing the process of automated extraction and purification of pathogen nucleic acid;

[0014] (3) After the chip is sealed with a sealing membrane, it is placed in an in-situ PCR instrument for amplification. After the reaction is completed, the detection results are observed under a fluorescence microscope.

[0015] Beneficial Effects

[0016] The core principle of the present invention is based on the nucleic acid extraction magnetic beads. The silanol or carboxyl groups on the surface can specifically bind to the nucleic acids in the solution through hydrogen bonds and electrostatic interactions. Therefore, nucleic acids can be directly and rapidly separated from complex biological systems, such as Figure 1As shown. The present invention successfully realizes the automation, integration and miniaturization of the system by changing the external magnetic field into a PCB electromagnetic coil combined with a small permanent magnet. At the same time, it also integrates the advantages of microfluidic chips. Through precisely designed microchannels and microchambers, the complex nucleic acid extraction process can be integrated into a small chip. Users only need simple sample loading and some basic operations to complete the entire nucleic acid extraction process, which has good market application prospects. The present invention can realize the automation, integration and miniaturization of nucleic acid samples from pretreatment to extraction and purification, with very little consumption of reagents and samples; high extraction efficiency, and high-purity products can be obtained; external interference and pollution are small, and it is more suitable for detection and diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is the extraction principle diagram of the present invention.

[0018] Figure 2 Schematic diagram of electromagnetic coil, microfluidic dimensions and layout.

[0019] Figure 3 Schematic diagram of the structure of the microfluidic chip.

[0020] Figure 4 Schematic diagram of the structure of the automated magnet control system.

[0021] Figure 5 Schematic diagram of magnetic bead manipulation.

[0022] Figure 6 Schematic diagram of automated nucleic acid purification.

[0023] Figure 7 It is a front view of the automated electromagnetic microfluidic chip of the present invention.

[0024] Figure 8 It is a side view of the automated electromagnetic microfluidic chip of the present invention.

[0025] Fig. 9 This is a bottom view of the automated electromagnetic microfluidic chip of the present invention. DETAILED DESCRIPTION

[0026] 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.

[0027] Example 1

[0028] like Figure 7-9As shown, this embodiment provides an automated electromagnetic microfluidic chip for nucleic acid purification, including a multi-channel microfluidic detection chip and an automated magnet control system located below the chip; the automated magnet control system includes a PCB electromagnetic coil and a plurality of permanent magnets placed above the PCB electromagnetic coil; the coil units of the PCB electromagnetic coil correspond one-to-one to the chamber unit positions of the multi-channel microfluidic detection chip.

[0029] In this embodiment, 4 holes are used as a unit. The microfluidic chip with 4 holes as a unit is called a single channel (detecting a pathogen nucleic acid), so multiple such units are multi-channel. The dimensions of the electromagnetic coil and microfluidic are as above. Figure 2 As shown, only 8 groups are drawn here, and the specific expansion will be based on actual conditions.

[0030] 1. The process of making a microfluidic chip with 4 holes as a unit in this embodiment includes the following steps:

[0031] 1.1 Silicon Wafer Production

[0032] (1) First, use AutoCAD software to draw the layout of the designed chip structure and save it in a file format that can be recognized by the lithography machine. Here, the small desktop maskless direct writing lithography system MicroWriter ML3 is used.

[0033] (2) Pretreatment: Use a nitrogen gun to clean impurity particles on the surface of a 4-inch silicon wafer.

[0034] (3) Spreading the film: Fix the pretreated silicon wafer in the center of the spreader, use 100 μm thick SU-83050 photoresist, and set the parameters of the spreader to 500 rpm, 10 s, 1300 rpm, 30 s.

[0035] (4) Pre-baking: Place the substrate after coating on a hot plate at 65°C for 5 minutes; then set the hot plate temperature to 95°C and keep it for 30 minutes.

[0036] (5) Exposure: Place the cooled silicon wafer in the photolithography machine, focus it, import the graphic file from the first step, and finally adjust the resistance sensitivity parameter to 2300 (mJ / cm 2 ).

[0037] (6) Post-baking: Place the exposed substrate on a hot plate at 65°C and keep it there for 1 minute. Then adjust the temperature of the hot plate to 95°C and keep it there for 5 minutes, and finally cool it down to room temperature.

[0038] (7) Development: The substrate sheets were placed in propylene glycol methyl ether acetate solution for development until no white precipitate appeared, and finally dried with a nitrogen gun.

[0039] (8) Hardening film: Place the developed silicon wafer on a hot plate at 150°C and bake for 20 minutes to evaporate the remaining SU-8 photoresist solvent and make the adhesion between the SU-8 photoresist on the substrate and the silicon wafer stronger, thus completing the preparation of the chip positive mold silicon wafer.

[0040] 1.2 Preparation of PDMS substrate

[0041] (1) PDMS prepolymer configuration: a certain mass of PDMS A liquid, the mass of curing agent B liquid is calculated according to the reagent mass of A liquid, the ratio is 10:1. After the PDMS and curing agent are mixed, stir them thoroughly with a stirring rod to form evenly distributed small bubbles in the plastic cup.

[0042] (2) Vacuum treatment of PDMS prepolymer: Place it in a vacuum drying pot and slowly evacuate it until all small bubbles are expelled.

[0043] (3) PDMS prepolymer pouring: Place the silicon wafer mold made in the first step horizontally in a plastic culture dish, and then pour the PDMS prepolymer from the middle of the silicon wafer positive mold until it completely covers the positive mold.

[0044] (4) PDMS prepolymer curing: Place the plastic petri dish in a 75°C oven and allow the PDMS prepolymer to stand in the oven for 3 hours. After the PDMS prepolymer is cured and polymerized, take it out of the oven.

[0045] (5) PDMS prepolymer demolding: Then gently separate the PDMS film from the silicon wafer.

[0046] (6) Punching holes in the PDMS substrate: Use a puncher with a hole diameter of 2 mm to vertically punch through holes at the opening of the microfluidic channel.

[0047] (7) PDMS substrate bonding: Place the PDMS substrate with the microchannel facing upwards and place it in a plasma cleaner together with the glass cover slip for 1 minute. Align the PDMS substrate and the glass substrate and bond them together. After bonding, place them in a vacuum drying oven at 65°C for 30 minutes to complete the preparation of a 4-well microfluidic chip.

[0048] 1.3 A more convenient production method

[0049] The production of a microfluidic chip usually requires steps 1.1 and 1.2. The whole process is time-consuming, and manual drilling can easily cause wear of the PDMS microfluidic chip and damage to the microfluidic channel. Therefore, in order to better produce a multi-channel microfluidic detection chip with 4-hole units as multiples, the method of pouring it into a CNC-machined aluminum alloy mold for curing can be used.

[0050] The details are as follows: by pouring PDMS into an aluminum alloy mold, and after the PDMS flows to cover the entire mold, a layer of plastic mold is attached on top, and a layer of weight is pressed on it for better shaping, and then heated at 100° for 3 hours to solidify. After solidification, a plasma bonding device is used to clean and bond the PDMS base and the glass substrate, and a high-throughput sample biological purification chip with a fine structure is formed after bonding. After a series of steps, a high-throughput sample biological microfluidic chip with uniform size and uniform reaction units is prepared. There are N identical reaction units on each chip, and the value of N refers to the manufactured mold. In this embodiment, N=16 channels are used, so that a large number of identical or different samples can be detected and analyzed in a centralized manner at one time, such as Figure 3 shown.

[0051] 2.Automated magnet control system

[0052] 2.1 Magnetic field source composition: PCB electromagnetic coil and permanent magnet, such as Figure 4 shown.

[0053] (1) PCB electromagnetic coil: It uses FR-4 insulating substrate material and a planar solenoid structure formed by stacking multiple layers of copper with a line width of 100 microns. The printed circuit board processing technology can achieve a high-precision, high-density coil matrix, and the matrix area covers the sensing area of ​​the microfluidic chip. The width of the formed coil is 2.64mm, and the spacing is controlled at 100 microns. According to the test, when the driving current of the two-layer coil with 5 turns is 400mA, it generates a magnetic field strength of about 0.5mT at a height of 1mm on the chip surface, which can drive (attract and move) a circular magnet with a diameter of 4mm and a thickness of 1mm.

[0054] (2) Permanent magnet: The diameter of the permanent magnet is 4mm, the height is 1mm, the material is N52, and the polarization direction is the Z-axis direction. N52 type permanent magnet is selected as the static magnetic field source to enhance the magnetic force. N52 refers to a grade of neodymium iron boron (NdFeB) permanent magnet, which is a material with persistent magnetism. It is processed into a cylindrical structure with a diameter of 4mm and a thickness of 1mm using a precision laser cutting machine. A permanent magnet is a magnet that generates a stable magnetic field. It can continuously generate a magnetic field in the absence of external current. When placed above the electromagnetic coil, the static magnetic field generated by the permanent magnet is superimposed on the electromagnetic field induced by the electromagnetic coil, thereby enhancing the overall magnetic field strength.

[0055] 2.2 Principle of precise control of magnetic beads:

[0056] When 400mA current is passed through coil 1, an induced electromagnetic field will be generated, attracting the magnet to move above the coil, and at the same time, the magnet will attract the magnetic beads in the microfluidic chip. According to this principle, a corresponding program is set up. For example, in order to mix the droplets evenly, the power of coil 1 and the nearest coil is turned on and off, and the effect is that the magnet oscillates back and forth, and the corresponding magnetic beads above oscillate back and forth in the droplets to achieve uniform mixing. Figure 5 As shown in the figure, if you want to allow the magnetic beads carrying nucleic acid to enter chamber 2 from chamber 1, set only coil 1 to be energized with a 200ms delay, at which time the magnet will drive the magnetic beads to break through the droplets; set only coil 2 to be energized with a 200ms delay, at which time the magnet will drive the magnetic beads into the channel between the two chambers; set only coil 3 to be energized, at which time the magnet will drive the magnetic beads into chamber 2.

[0057] The specific workflow of an automated electromagnetic microfluidic chip for nucleic acid purification in this embodiment is as follows: Figure 6 As shown, it can be mainly divided into three steps: adding reagents, automated magnetic bead nucleic acid extraction and amplification, and detection.

[0058] (1) Adding reagents: First, pre-fill 4 μL of aqueous elution reagent into the elution liquid chamber, then inject mineral oil into the lysis liquid chamber, tilt the chip to allow the mineral oil to flow through the bottom pipe until the first washing liquid chamber and the second washing liquid chamber are filled; finally, use a pipette to inject 4 μL of lysis liquid, 4 μL of the first washing liquid, and 4 μL of the second washing liquid into the corresponding chambers, and the oil seal makes each chamber independent and does not interfere with each other.

[0059] (2) Automated magnetic bead method for nucleic acid extraction: The sample is added to the lysis solution chamber, and the permanent magnet drives the magnetic beads to mix the reagent and let it stand for 8 minutes, waiting for the magnetic beads to fully adsorb the lysed nucleic acid. Next, the permanent magnet drives (attracts) the magnetic beads through the channel at the bottom and enters the first washing solution. Similarly, it can also enter the second washing solution. The magnetic beads are attracted by the magnet to shake back and forth for 1 minute to wash away contaminants such as proteins and salts. Then the magnetic beads are attracted to the elution solution chamber, mixed for 1 minute, and then let stand for 5 minutes to allow the nucleic acid to fully elute the aqueous elution reagent. Finally, the magnetic beads are attracted back to the second washing solution to complete the process of automated extraction and purification of pathogen nucleic acid.

[0060] (3) Amplification and detection: After the chip is sealed with a sealing membrane, it is placed in an in situ PCR instrument for amplification. After the reaction is completed, the detection results can be observed under a fluorescence microscope.

Claims

1. An automated electromagnetic microfluidic chip for nucleic acid purification, characterized in that: It includes a multi-channel microfluidic detection chip and an automated magnet control system located below the chip; the automated magnet control system includes a PCB electromagnetic coil and a plurality of permanent magnets placed above the PCB electromagnetic coil; the coil units of the PCB electromagnetic coil correspond one-to-one to the chamber unit positions of the multi-channel microfluidic detection chip.

2. The automated electromagnetic microfluidic chip according to claim 1, characterized in that: The chamber unit of the multi-channel microfluidic detection chip includes a lysate chamber, a first washing liquid chamber, a second washing liquid chamber and an elution liquid chamber, that is, a single channel.

3. The automated electromagnetic microfluidic chip according to claim 1, characterized in that: The multi-channel microfluidic detection chip is prepared by pouring PDMS into an aluminum alloy mold, attaching a plastic film to the surface and then heating and curing it, and using a plasma bonding device to clean and bond the PDMS base and the glass substrate after molding, thereby obtaining a multi-channel microfluidic detection chip.

4. The automated electromagnetic microfluidic chip according to claim 1, characterized in that: The PCB electromagnetic coil adopts FR-4 insulating substrate material and is a planar solenoid structure formed by stacking multiple layers.

5. The automated electromagnetic microfluidic chip according to claim 1, characterized in that: The material of the permanent magnet is N52.

6. A method for using the automated electromagnetic microfluidic chip for nucleic acid purification according to any one of claims 1 to 5, comprising the following steps: (1) firstly, pre-filling the aqueous elution reagent into the elution liquid chamber, then injecting mineral oil into the lysis liquid chamber, tilting the chip multi-channel microfluidic detection chip to allow the mineral oil to flow through the bottom pipeline until the first washing liquid chamber and the second washing liquid chamber are filled; finally, using a pipette gun to inject the lysis liquid, the first washing liquid, and the second washing liquid into the corresponding chambers; (2) adding the sample to the lysate chamber, driving the magnetic beads to mix the reagent by a permanent magnet and letting it stand, waiting for the magnetic beads to fully adsorb the lysed nucleic acid; then driving the magnetic beads through the channel at the bottom to enter the first washing liquid chamber, and similarly enter the second washing liquid chamber, and attracting the magnetic beads to vibrate back and forth by the magnet; then attracting the magnetic beads to the elution liquid chamber, mixing and letting it stand, so that the nucleic acid is fully eluted in the aqueous elution reagent; finally, attracting the magnetic beads back to the second washing liquid chamber, completing the process of automated extraction and purification of pathogen nucleic acid; (3) After the chip is sealed with a sealing membrane, it is placed in an in-situ PCR instrument for amplification. After the reaction is completed, the detection results are observed under a fluorescence microscope.

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