CN117821227A - Microfluidic-based cracking chip and preparation method and application thereof - Google Patents

Abstract

The invention provides a microfluidic-based cracking chip, and belongs to the technical field of microfluidics. The cracking chip comprises a chip upper layer, a chip middle layer and a chip lower layer from top to bottom in sequence. The chip upper layer is provided with a sample inlet, a cracking runner, a purification chamber, a plurality of washing chambers, a detection chamber and a sample outlet which penetrate through the chip upper layer and are sequentially communicated. The upper chip layer is fixedly connected with the middle chip layer, and the lower chip layer is connected with the upper chip layer and the middle chip layer through clamping; and an ultrasonic wave generating device is arranged on the upper surface of the lower layer of the chip. According to the application, the ultrasonic wave generating device is introduced into the chip and is matched with the magnetic beads for use, the ultrasonic wave can generate strong mechanical force, so that the cell membrane can be effectively cracked to release biomolecules in cells, and the chip has high cracking efficiency, uniformity and simplicity in operation.

Description

Microfluidic-based cracking chip and preparation method and application thereof

Technical Field

The invention relates to the technical field of microfluidics, in particular to a microfluidic-based cracking chip.

Background

The living environment cannot be boiled, the water quality safety is closely related to the life safety of people, and the exceeding of escherichia coli in water can cause harm to the health of human bodies, and can cause intestinal diseases such as vomiting, diarrhea and the like. The water quality escherichia coli detector can detect the content of colonies such as escherichia coli, fecal (heat-resistant) escherichia coli, enterococcus and pseudomonas aeruginosa, so as to ensure the water quality safety. In tap water, total coliform, heat-resistant coliform and Escherichia coli cannot be detected, and the total colony count cannot exceed 100CFU/mL. The mainstream detection method still depends on the traditional culture method, and although the detection sensitivity of the method is high, the following two problems still exist: firstly, long incubation time is required, so that the detection time is too long; secondly, multi-step reaction is needed, so that sample addition is needed manually, and the probability of cross contamination is increased. The detection of escherichia coli has important significance in the fields of food safety, water quality monitoring and medical treatment, and can help to protect public health and prevent disease transmission.

In view of the above-described shortcomings of the detection methods, the present inventors aimed at developing microfluidic-based cleavage chips of the present invention through long-term studies and practices.

Disclosure of Invention

In view of the above-mentioned shortcomings of the prior art, a main object of the present invention is to provide a microfluidic-based lysis chip, in which an ultrasonic wave generating device is introduced into the chip and used in combination with magnetic beads, the ultrasonic wave can generate a strong mechanical force, which helps to effectively lyse cell membranes and release biomolecules in cells, so that the efficiency of lysis, uniformity and operation are high, and in addition, the types of ultrasonic waves and magnetic beads generated are adjustable, so that the chip can be optimized and adjusted according to experimental requirements and sample properties, so that the chip has customizable properties.

To achieve the above and other related objects, the present invention is achieved by comprising the following technical solutions.

The invention provides a microfluidic-based cracking chip, which sequentially comprises a chip upper layer, a chip middle layer and a chip lower layer from top to bottom;

the chip upper layer is provided with a sample inlet, a cracking flow channel, a purification chamber, a plurality of washing chambers, a detection chamber and a sample outlet which penetrate through the chip upper layer and are sequentially communicated;

the upper chip layer is fixedly connected with the middle chip layer, and the lower chip layer is connected with the upper chip layer and the middle chip layer through clamping; and an ultrasonic wave generating device is arranged on the upper surface of the lower layer of the chip.

In some preferred embodiments, the ultrasonic wave generating device is a piezoelectric ceramic ultrasonic transducer, which is externally connected to a power supply.

In some preferred embodiments, the upper surface of the lower layer of the chip is also provided with two piezoelectric ceramic electrodes which are oppositely arranged, and the two piezoelectric ceramic electrodes are respectively externally connected with a power supply.

In some preferred embodiments, the material of the piezoceramic electrode is at least one selected from Cr or Au.

In some preferred embodiments, the upper surface of the lower layer of the chip is also provided with heating means;

the ultrasonic wave generating device is arranged below the cracking flow channel, and the heating device is arranged below the purifying chamber, the washing chambers and the detecting chamber.

In some preferred embodiments, the heating device is a heated metal disk that is externally connected to a power source.

In some preferred embodiments, the lower chip layer is in clamping connection with the upper chip layer and the middle chip layer by a clamp;

the fixture comprises a fixture upper cover and a fixture base which are detachably connected, wherein the fixture base is provided with a fixing groove, and the shape of the fixing groove is matched with the lower layer of the chip;

the fixture upper cover is provided with a hollowed-out window, and the middle layer of the chip and the upper layer of the chip are exposed out of the fixture from the hollowed-out window.

In some preferred embodiments, the sample injection hole is located at one side of the upper layer of the chip, the cracking channel is arranged in a zigzag or serpentine shape extending from one side of the upper layer of the chip to the other side, and one end of the cracking channel is communicated with the sample injection hole, and the other end of the cracking channel is communicated with the purification chamber.

In some preferred embodiments, the purification chamber and the several washing chambers are co-linear.

In some preferred embodiments, the material of the upper layer of the chip is selected from polydimethylsiloxane or COC plastic or polymethyl methacrylate.

In some preferred embodiments, the middle layer of the chip is a Si sheet and the material of the lower layer of the chip is glass.

In some preferred embodiments, the on-chip layer and the on-chip layer are connected by plasma bonding.

In some preferred embodiments, the diameter of the sample inlet is 0.5-2 mm, the thickness of the upper layer of the chip is 2-5 mm, the length of the cracking flow channel is 30-60 cm, the width of the cracking flow channel is 50-200 μm, and the diameters of the purification chamber and the washing chamber are 3-5 mm.

The second aspect of the invention provides a preparation method of the microfluidic-based cracking chip, which comprises the following steps: 1) Providing a middle layer of the chip;

2) Spin-coating SU-8 photoresist on the middle layer of the chip, wherein the spin-coating thickness is 100-400 mu m;

3) Patterning a high resolution photomask containing a channel design by exposing it to ultraviolet light, exposing and baking, and immersing in SU-8 developer to complete the channel pattern;

4) Mixing the material of the upper layer of the chip with a curing agent, pouring the mixture on the channel pattern, removing redundant SU-8 photoresist after curing to obtain the upper layer of the chip, and obtaining the upper layer of the chip and the middle layer of the chip which are fixedly connected through plasma treatment;

5) Providing a lower chip layer, and fixing an ultrasonic wave generating device on the lower chip layer;

6) And providing a clamp, and connecting the lower chip layer with the upper chip layer and the middle chip layer through clamping.

The third aspect of the invention provides the application of the microfluidic-based lysis chip in bacterial or cell lysis.

The fourth aspect of the present invention provides a method for using the microfluidic-based chip, comprising the following steps:

1) Preparing a sample: providing obtained 10 ⁵ ～10 ⁷ Bacterial liquid or cell liquid with CFU/ml concentration;

2) Providing a cracking liquid and a washing liquid;

3) Preparing biological functionalized magnetic beads, and covalently coupling streptavidin on the surfaces of the magnetic beads;

4) A magnetic block is placed at the bottom of the last washing chamber, so that the magnetic beads are ensured to be precipitated in the last washing chamber;

5) Adding a sample and biological functional magnetic beads into a sample injection hole, and respectively adding a cracking liquid, a purifying liquid and a washing liquid into a cracking flow channel, a purifying chamber and a plurality of washing chambers;

6) Starting the ultrasonic wave generating device to complete the bacterial lysis and obtain target molecules.

As described above, the microfluidic-based cracking chip of the present invention has the following advantages:

1) Cell disruption, DNA extraction, purification and preservation can be achieved by continuous on-chip processing, and ultrasonic waves can generate intense mechanical forces that help to effectively lyse cell membranes and release biomolecules within cells. The use of the magnetic beads can enhance the stirring and mixing effects and improve the cracking efficiency.

2) The use of ultrasonic waves in combination with magnetic beads ensures that the lysis and mixing in the sample is uniform, resulting in more consistent experimental results.

3) The operation of ultrasonic combined magnetic bead chip is relatively simple, and only the sample and the magnetic beads are mixed and then placed in the chip, so that a complex microfluidic system is not needed. This may reduce the technical requirements and operational complexity of the experiment.

4) Ultrasonic lysis can generally be accomplished in a short period of time, thereby improving the rapidity of the experiment. This is particularly important for experiments requiring high throughput processing.

5) The method of ultrasound and magnetic bead binding is generally applicable to a variety of sample types and cell types, making them more versatile.

6) The ultrasonic wave pyrolysis and the use of the magnetic beads are adjustable, and can be optimized and adjusted according to specific experimental requirements and sample properties, so that the experimental customization is improved.

Drawings

FIG. 1 shows an exploded view of a cleavage chip.

Fig. 2 shows a top view (in a transparent state) of the upper chip layer of the cleaved chip.

Fig. 3 shows a top view of the lower layer of the chip as a cleaved chip.

Fig. 4 shows an exploded view of the clamp.

FIG. 5 shows a schematic diagram of the chip in a chip-lysing application.

The reference numerals in fig. 1 to 5 are as follows:

1. chip upper layer

11. Sample injection hole

12. Cracking runner

13. Purification chamber

14. Washing chamber

15. Detection chamber

16. Sample outlet

2. Middle layer of chip

3. Lower layer of chip

4. Ultrasonic wave generating device

5. Heating device

6. Clamp

61. Clamp upper cover

611. Hollowed-out window

62. Clamp base

621. Fixing groove

7. Magnetic bead

Detailed Description

Further advantages and effects of the present invention will become apparent to those skilled in the art from the description given herein below, by way of specific examples.

Please refer to fig. 1 to 5. It should be understood that the structures, proportions, sizes, etc. shown in the drawings are for illustration purposes only and should not be construed as limiting the invention to the extent that it can be practiced, since modifications, changes in the proportions, or otherwise, used in the practice of the invention, are not intended to be critical to the essential characteristics of the invention, but are intended to fall within the spirit and scope of the invention. Also, the terms such as "upper," "lower," "left," "right," "middle," and "a" and the like recited in the present specification are merely for descriptive purposes and are not intended to limit the scope of the invention, but are intended to provide relative positional changes or modifications without materially altering the technical context in which the invention may be practiced.

Example 1

Referring to fig. 1 to 5, the invention provides a microfluidic-based cracking chip, which sequentially comprises a chip upper layer 1, a chip middle layer 2 and a chip lower layer 3 from top to bottom. Referring to fig. 2, the upper chip layer 1 is provided with a sample inlet 11, a cleavage flow channel 12, a purification chamber 13, a plurality of washing chambers 14, a detection chamber 15 and a sample outlet 16 which penetrate through the upper chip layer 1 and are sequentially communicated. The chip upper layer 1 is fixedly connected with the chip middle layer 2, the chip lower layer 3 is connected with the chip upper layer 1 and the chip middle layer 2 through clamping, and an ultrasonic wave generating device 4 is arranged on the upper surface of the chip lower layer 3.

Specifically, continuous on-chip processing can be performed by the above-described structural arrangement to achieve cell disruption, DNA extraction, purification, and preservation, bacterial liquid or cell liquid to be lysed is mixed with magnetic beads, fed into the chip from the sample inlet 11, and mechanically stirred or vibrated together with the sample by using mechanical force generated by high-frequency ultrasonic oscillation, thereby disrupting the cells to release their internal substances. And then the target molecules such as DNA, RNA or protein functional groups can be adsorbed on the surfaces of the magnetic beads, so that the target molecules can be obtained through separation. The chip has simple structure, combines the mechanical force of ultrasonic wave and the stirring effect of magnetic beads, and can improve the cracking efficiency and the uniformity of the sample.

In a specific example, the ultrasonic wave generating device 4 is a piezoelectric ceramic ultrasonic transducer, and the piezoelectric ceramic ultrasonic transducer is externally connected with a power supply. The piezoelectric ceramic ultrasonic transducer suitable for the chip has the diameter of 5-20 mm and the thickness of 1-10 mm; the frequency of the piezoelectric ceramic ultrasonic transducer suitable for the chip is 50-70MHz.

In one embodiment, the lower layer of the chip is provided with a magnet moving device for controlling the movement of the magnetic beads so as to promote the combination of the magnetic beads and target molecules.

In one embodiment, the material of the piezoelectric ceramic electrode is selected from lead zirconate titanate (PZT). By applying voltage to the electrodes, an electric field is generated between the electrodes, and by controlling the strength of the electric field, the vibration of the magnetic beads is realized.

In a specific example, the upper surface of the lower chip layer 3 is further provided with heating means 5. The heating device 5 is a heating metal disc, and the heating metal disc is externally connected with a power supply. The heating metal disc is a conventional wire heating disc, and the size of the heating metal disc is matched with that of the chip.

More specifically, referring to fig. 3, the ultrasonic wave generating device 4 is disposed below the cleavage flow path 12, and the two piezoceramic electrodes are also disposed below the cleavage flow path 12, and the heating device 5 is disposed below the purification chamber 13, the plurality of washing chambers 14, and the detection chamber 15. The magnetic bead vibration and the ultrasonic vibration are provided by the combined action of the ultrasonic wave generating means 4 and the electromagnetic field to complete cell disruption.

In one embodiment, referring to fig. 1 and 4, the lower chip layer 3 is clamped to the upper chip layer 1 and the middle chip layer 2 by a clamp 6. The fixture 6 comprises a fixture upper cover 61 and a fixture base 62 which are detachably connected, the fixture base 62 is provided with a fixing groove 621, the shape of the fixing groove 621 is matched with that of the chip lower layer 2, the fixture upper cover 61 is provided with a hollowed-out window 611, and the chip middle layer 2 and the chip upper layer 1 are exposed outside the fixture 6 from the hollowed-out window 611. More specifically, the shape of anchor clamps and the shape adaptation of chip, in this case rectangle, set up through-hole or screw hole in the four corners of rectangle to cooperate bolt or screw to carry out detachable connection, the centre gripping is stable and convenient operation.

In some embodiments, 1) referring to fig. 2, the sample injection hole 11 is located at one side of the upper chip layer 1, the cleavage channel 12 is arranged in a zigzag or serpentine shape extending from one side of the upper chip layer 1 to the other side, and one end of the cleavage channel 12 is connected to the sample injection hole 11, and the other end is connected to the purification chamber 13. 2) The purification chamber 13 and the several washing chambers 14 are located in the same line. Reasonable structural layout, and is beneficial to the processing and the use of chips.

In some embodiments, the diameter of the sample inlet 11 is 0.5-2 mm, the thickness of the upper layer 1 on the chip is 2-5 mm, the length of the cleavage flow channel 12 is 30-60 cm, the width of the cleavage flow channel 12 is 50-200 μm, the depth of the cleavage flow channel 12 is 100-400 μm, and the diameters of the purification chamber 13 and the washing chamber 14 are 3-5 mm. Preferably, the cleavage flow channel 12 has a depth of 100 to 200 μm and a width of 80 to 150 μm, and the size range is adapted to a piezoelectric ceramic ultrasonic transducer having a frequency of 1 to 5 MHz. In addition, the length of the lysis channel 12 should be ensured within a range of 30 to 60cm to ensure complete lysis of cells or bacteria.

In some embodiments, the material of the upper chip layer 1 is selected from polydimethylsiloxane or COC plastic or polymethyl methacrylate. The middle chip layer 2 is a Si sheet (silicon wafer), the lower chip layer 3 is made of glass, and the upper chip layer 1 and the middle chip layer 2 are connected through plasma bonding.

Example 2

The preparation method of the microfluidic-based cracking chip comprises the following steps:

1) Providing a 3-inch silicon wafer, and cleaning and drying;

2) Spin-coating with 100 μm SU-8 (NANO SU-8 50,MicroChem,Newton,USA) Photoresist (PR) layer. Then soft baked at 80 c for 10 minutes and then baked at 110 c for another 30 minutes to evaporate the solvent on the silicon wafer. When the silicon wafer reached room temperature, a new SU-8 layer (100 μm thick) was spun and soft baked in the same way. After three consecutive deposition steps, a 300 μm thick PR layer was obtained on top of the silicon wafer.

3) Subsequently, the high resolution photomask containing the channel design is patterned by exposure to Ultraviolet (UV) light. After post exposure bake, the wafer is immersed in SU-8 developer to complete the channel pattern. The remaining crosslinked SU-8PR was silicone polymer forming the raised mold.

4) PDMS (Sylgard 184,Dow Corning,Midland,USA) was prepared at 10:1 and a curing agent, and poured onto the patterned wafer. The polymer mixture was cured at 65℃for more than 2 hours. After curing, the mold is removed and the flat-ended dispensing needle is used to drill the attachment holes into the device. The channels are then bonded to the silicon wafer substrate by oxygen plasma treatment.

5) The lower chip layer 3 is provided as a glass sheet and is grooved, and then two piezoceramic transducers with frequencies of 50-70MHz are fixed in the grooves and a metal disk heater is placed or fixed in the grooves.

6) A clamp 6 as shown in fig. 4 is provided to connect the lower chip layer 3 with the upper chip layer 1 and the middle chip layer 2 by clamping.

Example 3

The using method of the microfluidic-based cracking chip comprises the following steps:

1) Preparing a sample: coli was incubated at 37 ℃, shaken in a water bath at 120RPM, and the suspension was monitored for changes in optical density at 600nm (OD 600) using a DU-800 spectrophotometer (Beckman Instruments, inc., fullerton, CA, USA) and its growth was followed. Cells were grown to log phase harvest, washed twice in 1×pbs (137mM NaCl,2.7mM KCl,4.3mM Na2HPO4 · 7H2O,1.5mM KH2PO4) and at an average cell density of 5×10 ⁷ CFU/The mL was resuspended in 1 XPBS. The stock suspension was stored cold and all experiments were completed within two hours after initial sample preparation. A cell suspension with a volume of 50 μl was pumped into the microfluidic system using a syringe pump.

2) Preparing magnetic beads: injection methods are used to fill the microchannels with sol-gel-bead matrices. The sol was prepared by hydrolysis of 27% v/v Tetraethoxysilane (TEOS) (Sigma-Aldrich, st.Louis, MO, USA) in water by addition of 0.1% v/v HNO, heated to 60℃for 10 minutes, then heated at 80℃for 60 minutes, and stirred at 200 rpm. Silica microbeads having a diameter of 5 μm were added to 1mL of the sol at a concentration of 200mg of beads prior to gelation. Gelation of the sol is carried out by raising the temperature to 100 ℃ to effect condensation under acidic conditions. The use of silica microbeads having a diameter of 5 μm minimizes sedimentation problems when preparing suspensions, as compared to suspensions having larger silica microbead diameters. The resulting sol-gel silica microbead matrix binds DNA/RNA by electrostatic interactions. The sol-gel silica bead matrix is hydrophilic in that it is formed by acid catalyzed sol condensation. Silica microbeads provide a large surface area for selective binding of DNA, while sol-gels are used as silica-based water for immobilizing microbeads in place during device operation. The sol-gel-microbead matrix was introduced into the channel under pressure using a sample injection method, the chamber outlet was blocked by 90% to allow some liquid to flow out while confining the majority of the beads in the channel. After filling, the chamber was dried at 120 ℃ for 24 hours.

3) Cracking process

(1) Injecting the sample into the serpentine region, then stopping (static) and driving the piezoelectric ceramic transducer;

(2) The sample continuously flows (flows) through the serpentine region during the lysis process. A composite signal generator and a radio frequency power amplifier are used to drive the piezoceramic transducer. Under the duty ratio of 100%, the input power is 0-200 mW, and the sample flow is 10-25 mu L/min, so that the cracking efficiency is improved to the maximum extent, and excessive heat and acoustic cavitation are avoided.

4) Cracking verification

Lysis efficacy was quantified by measuring ATP released from sonicated samples and comparing it to ATP of untreated samples. Firefly luciferase-based ATP assays were performed in the form of 96-well microwell plates with a limit of detection of 0.1pmol of ATP. Fluorescence value measurements were performed using a Varioskan LUX multifunctional microplate reader.

The extraction process was initiated by rinsing the microchannel with MeOH to activate the silica. The microchannel was rinsed with TE (10mM Tris,1mM EDTA), titrated to pH 7.6 with HCl, and 6M guanidine hydrochloride. All solutions were prepared in 18 M.OMEGA.cm water. The sample was loaded in 6M guanidine hydrochloride using 25. Mu.L of DNA (250 fmol) and flowed through the microchannel at a rate of 2. Mu.L/min. The washing step was performed using 25 μl of 80% 2-propanol. This removes any excess guanidine hydrochloride and contaminants from the microchannels. DNA was eluted from the solid phase by flowing TE buffer through the micro-channels. The loading, wash and eluate were collected in microcentrifuge tubes and analyzed using DNAQF DNA quantitative fluorometry kit (Sigma-Aldrich, st.Louis, MO, USA).

The above embodiments are merely illustrative of the principles of the present invention and its effectiveness, and are not intended to limit the invention. Modifications and variations may be made to the above-described embodiments by those skilled in the art without departing from the spirit and scope of the invention. Accordingly, it is intended that all equivalent modifications and variations of the invention be covered by the claims, which are within the ordinary skill of the art, be within the spirit and scope of the present disclosure.

Claims (10)

1. The micro-fluidic-based cracking chip is characterized by comprising a chip upper layer (1), a chip middle layer (2) and a chip lower layer (3) from top to bottom in sequence;

the chip upper layer (1) is provided with a sample injection hole (11), a cracking runner (12), a purification chamber (13), a plurality of washing chambers (14), a detection chamber (15) and a sample outlet hole (16) which penetrate through the chip upper layer (1) and are sequentially communicated;

the chip upper layer (1) is fixedly connected with the chip middle layer (2), and the chip lower layer (3) is connected with the chip upper layer (1) and the chip middle layer (2) through clamping;

and the upper surface of the chip lower layer (3) is provided with an ultrasonic wave generating device (4).

2. The microfluidic-based cracking chip according to claim 1, wherein the ultrasonic wave generating device (4) is a piezoelectric ceramic ultrasonic transducer, and the piezoelectric ceramic ultrasonic transducer is externally connected with a power supply;

and/or, the upper surface of the chip lower layer (3) is also provided with two piezoelectric ceramic electrodes which are oppositely arranged, and the two piezoelectric ceramic electrodes are respectively externally connected with a power supply.

3. The microfluidic-based chip according to claim 2, wherein the material of the piezoceramic electrode is at least one selected from Cr or Au.

4. Microfluidic based lysis chip according to claim 1, characterized in that the upper surface of the chip lower layer (3) is further provided with a heating device (5);

the ultrasonic wave generating device (4) is arranged below the cracking flow channel (12), and the heating device (5) is arranged below the purifying chamber (13), the washing chambers (14) and the detecting chamber (15).

5. The microfluidic-based chip according to claim 4, wherein the heating device (5) is a heating metal plate, and the heating metal plate is externally connected with a power supply.

6. Microfluidic based lysis chip according to claim 1, characterized in that the lower chip layer (3) is in clamping connection with the upper chip layer (1) and the middle chip layer (2) by means of a clamp (6);

the fixture (6) comprises a fixture upper cover (61) and a fixture base (62) which are detachably connected, the fixture base (62) is provided with a fixing groove (621), and the shape of the fixing groove (621) is matched with that of the chip lower layer (2);

the fixture upper cover (61) is provided with a hollowed-out window (611), and the middle chip layer (2) and the upper chip layer (1) are exposed out of the fixture (6) from the hollowed-out window (611).

7. The microfluidic based lysis chip according to any of claims 1 to 6, comprising at least one of the following technical features:

a1 The sample injection hole (11) is positioned at one side of the chip upper layer (1), the cracking channel (12) is in a zigzag shape or a serpentine shape and extends from one side of the chip upper layer (1) to the other side, one end of the cracking channel (12) is communicated with the sample injection hole (11), and the other end is communicated with the purification chamber (13);

a2 -said purification chamber (13) and several said washing chambers (14) are positioned in the same line;

a3 A material of the upper chip layer (1) is selected from polydimethylsiloxane or COC plastic or polymethyl methacrylate;

a4 The middle layer (2) of the chip is a Si sheet, and the lower layer (3) of the chip is made of glass;

a5 The chip upper layer (1) and the chip middle layer (2) are connected through plasma bonding;

a6 The diameter of the sample injection hole (11) is 0.5-2 mm, the thickness of the upper chip layer (1) is 2-5 mm, the length of the cracking runner (12) is 30-60 cm, the width of the cracking runner (12) is 50-200 mu m, and the diameters of the purification chamber (13) and the washing chamber (14) are 3-5 mm.

8. The method for preparing the microfluidic-based cracking chip according to any one of claims 1 to 7, comprising the following steps:

1) Providing a chip middle layer (2);

2) Spin-coating SU-8 photoresist on the middle layer (2) of the chip, wherein the spin-coating thickness is 100-400 mu m;

3) Patterning a high resolution photomask containing a channel design by exposing it to ultraviolet light, exposing and baking, and immersing in SU-8 developer to complete the channel pattern;

4) Mixing the material of the upper layer of the chip with a curing agent, pouring the mixture on a channel pattern, removing redundant SU-8 photoresist after curing to obtain the upper layer (1) of the chip, and obtaining the upper layer (1) of the chip and the middle layer (2) of the chip which are fixedly connected through plasma treatment;

5) Providing a chip lower layer (3), and fixing an ultrasonic wave generating device (4) on the chip lower layer (3);

6) And providing a clamp (6), and connecting the lower chip layer (3) with the upper chip layer (1) and the middle chip layer (2) through clamping.

9. Use of a microfluidic based lysis chip according to any of claims 1 to 7 for bacterial lysis or cell lysis.

10. The method for using a microfluidic-based chip according to any one of claims 1 to 7, comprising the steps of:

1) Preparing a sample: providing obtained 10 ⁵ ～10 ⁷ Bacterial liquid or cell liquid with CFU/ml concentration;

2) Providing a lysate, a purified solution and a washing solution;

3) Preparing biological functionalized magnetic beads, and covalently coupling streptavidin on the surfaces of the magnetic beads;

4) A magnetic block is placed at the bottom of the last washing chamber, so that the magnetic beads are ensured to be precipitated in the last washing chamber;

5) Adding a sample and biological functional magnetic beads into a sample injection hole (11), and respectively adding a cracking liquid, a purifying liquid and a washing liquid into a cracking flow channel (12), a purifying chamber (13) and a plurality of washing chambers (14);

6) Starting the ultrasonic wave generating device to complete the bacterial lysis and obtain target molecules.