Cell lysis microfluidic biochip and method based on surface acoustic wave principle

Abstract

The invention belongs to the technical field of microfluidics, and particularly relates to a cell lysis microfluidic biochip based on a surface acoustic wave principle and a method. Wherein the interdigital electrode comprises bus bars and an electrode pair between the bus bars, the electrode pair comprises electrode finger strips and a suspension electrode structure, the electrode finger strips are fixed on the front bus bar and the rear bus bar, the suspension finger strips are arranged in door-shaped finger strips of the suspension electrode structure, and the front end, the middle end and the rear end of a micro-channel plate of the micro-channel are respectively provided with an inlet, a cracking chamber and an outlet. A micro-column baffle is arranged at the end, close to the outlet flow channel, of the cracking chamber, and magnetic particles are placed in the cracking chamber; the liquid drops are driven to move at a high speed through the acoustic microflow effect, cells in the liquid drops are driven to move and collide with particles rapidly, rapid and efficient cell lysis is achieved, and therefore the device is suitable for lysis analysis and biochemical detection of a small number of cell samples.

Description

Technical Field

[0001] The invention belongs to the field of microfluidic technology, and specifically relates to a cell lysis microfluidic biochip and method based on the surface acoustic wave principle. Background Art

[0002] Surface acoustic wave (SAW) is a new technology that can realize on-chip operation. Since the formation and propagation process of surface acoustic wave carries energy and momentum, and the energy and momentum of the wave can be manipulated through various designs, surface acoustic wave (SAW) has been widely used in the field of microfluidics due to its non-contact and biocompatibility. It has broad application prospects in biomedicine, diagnostics and other fields and is a widely used new technology.

[0003] In the past decade, biochips based on the combination of surface acoustic wave and microfluidic technology have attracted widespread attention and development since their emergence, and have developed rapidly. Researchers have designed and developed microfluidic devices based on SAW mixing, atomization, particle manipulation, droplet drive, particle enrichment, cell culture and other functions, all of which have shown great potential in different fields.

[0004] As a powerful operation and detection platform, microfluidic technology can be used to realize complex cell operations, sample purification, biochemical reaction simulation and various detections on a chip of several square centimeters (Microfluidic Chip), and also provides a new tool for biological sample pretreatment. This microchip system provides an important method for realizing small sample operations, reducing the amount of expensive biochemical reagents, improving detection accuracy and speed, and especially reducing the number of samples in biochemical sample processing. It also provides an important new method for the miniaturization and integration of biochemical inspection devices and equipment.

[0005] Micro-nano materials can be precisely manipulated by means of acoustic field structure design and sound wave intensity adjustment, thus giving rise to the technology corresponding to the "optical tweezers" technology, called "acoustic tweezers". As the name suggests, SAW acoustic tweezers technology can achieve a series of functions such as cell driving, transporting, enriching, manipulating and even lysing at the micro-nano scale.

[0006] The traditional method of cell lysis and separation and purification requires the use of special equipment, and the cell lysis is inefficient and uneven, the intracellular substances extracted after cell lysis are impure or the extraction rate is low, the operation cost is high, the steps are complicated, and the separation efficiency is low, which greatly limits the cell lysis and purification effect. At the same time, the traditional cell lysis method also has problems such as large sample requirements, slow cell lysis speed and poor effect, and certain potential hazards to operators. Summary of the invention

[0007] In order to overcome the above problems, the present invention provides a cell lysis microfluidic biochip and method based on the surface acoustic wave principle, as well as a method for fast, efficient and easy-to-control lysis treatment of trace cell samples based on surface acoustic waves with good mechanical properties and stable transmission characteristics. The present invention applies surface acoustic waves (SAW) and acoustic capture technology to microliter droplets, drives the droplets to move at high speed through the acoustic microfluidic effect, drives the cells and particles in the droplets to move and collide quickly, so as to achieve fast and efficient cell lysis, so as to be suitable for lysis analysis and biochemical detection of a small amount of cell samples.

[0008] A cell lysis microfluidic biochip based on the surface acoustic wave principle, comprising an interdigital electrode 1, a microfluidic channel 2 and a lithium niobate piezoelectric substrate 3, wherein the microfluidic channel 2 and the interdigital electrode 1 are fixed on the lithium niobate piezoelectric substrate 3 in sequence from left to right;

[0009] The interdigitated electrode 1 includes a plurality of electrode pairs and a bus bar 16, wherein a plurality of electrode pairs are arranged between the bus bars 16, and the electrode pair includes two left and right electrode fingers 11 and a suspended electrode structure 15 located between the electrode fingers 11, wherein the bottom of the electrode fingers 11 is fixed on the lithium niobate piezoelectric substrate 3, and the rear end of the left electrode finger 11 is fixed on the rear end bus bar 16, and the front end of the right electrode finger 11 is fixed on the front end bus bar 16, and the suspended electrode structure 15 includes a gate-type finger 13 and a suspended finger 12, wherein the gate-type finger 13 is provided with uniformly distributed suspended fingers 12, and the bottoms of the gate-type finger 13 and the suspended finger 12 are both fixed on the lithium niobate piezoelectric substrate 3;

[0010] The microfluidic channel 2 includes a microfluidic plate, magnetic particles 26 and a microcolumn baffle 27, wherein the front, middle and rear ends of the microfluidic plate are respectively provided with an inlet 21, a lysis chamber 23 and an outlet 25, wherein the inlet 21 is connected with the lysis chamber 23 through an inlet channel 22, and the lysis chamber 23 and the outlet 25 are connected through an outlet channel 24, and a microcolumn baffle 27 is provided at one end of the lysis chamber 23 close to the outlet channel 24, and a plurality of magnetic particles 26 are placed in the lysis chamber 23.

[0011] The micro-column baffle 27 includes a row of multiple micro-columns arranged in an arc shape, and is placed at one end of the lysis chamber 23 close to the outlet flow channel 24 and fixed in the lysis chamber 23 .

[0012] The inlet 21 and the outlet 25 are both through holes.

[0013] The bus bar 16 includes a long bar and a short bar, wherein the short bar is fixed outside the right end of the long bar.

[0014] A method for realizing cell lysis by using a cell lysis microfluidic biochip based on the surface acoustic wave principle and using surface acoustic wave and microfluidic technology comprises the following steps:

[0015] Step S1-1: Connect a power source to one end of the short strip of the two bus bars 16 on the interdigital electrode 1, i.e., the input end, and simultaneously introduce the liquid to be lysed into the microchannel 2 through the inlet 21;

[0016] Step S1-2: After the interdigital electrodes 1 are energized, surface acoustic waves are generated on the lithium niobate piezoelectric substrate 3 and propagate toward the microchannel 2;

[0017] Step S1-3: The sound waves drive the cells in the liquid to be lysed in the microfluidic channel 2 to collide rapidly with the magnetic particles 26, thereby achieving cell lysis; wherein the liquid to be lysed flows into the lysis chamber 23 through the inlet 21 and the inlet channel 22. The process of introducing the liquid is continuous, and the unlysed cells cannot pass through the microcolumn baffle 27 composed of micro-cylinders. The contents after cell lysis will continue to flow out at the outlet 25 along with the liquid through the outlet channel 24, thereby achieving continuous cell lysis and timely collection of lysis products.

[0018] The method for manufacturing the interdigital electrode 1 includes the following contents:

[0019] Step 1: Using the lithium niobate piezoelectric substrate 3 as a substrate, a Cr film is deposited on the substrate by a thermal evaporation deposition apparatus, and then an Au film is deposited on the Cr film;

[0020] Step 2, covering the entire surface of the film after the evaporation in step 1 with photoresist, and then placing the film with the photoresist in a spin coater, and running the spin coater at two speeds, low speed and high speed, respectively, wherein the low speed stage is 1000 rpm for 5 seconds, and the high speed stage is 5500 rpm for 30 seconds, to obtain a film after photoresist is evenly coated;

[0021] Step 3: Place the coated film on a hot plate and bake it. The hot plate temperature is set to 110°C and the baking time is 2 minutes.

[0022] Step 4, using an exposure agent to expose the heated film, and after the exposure is completed, the film is placed on a hot plate for baking, the hot plate temperature is set to 100° C., and the baking time is 80 seconds to fix the connection between the photoresist and the film;

[0023] Step 5: Take 100 mL of KMPPD238-II positive photoresist developer and 40 mL of deionized water and mix them evenly. Place the baked film in the mixed solution and develop for 50 seconds. Then, rinse the film twice with deionized water, each time for 20 seconds to remove the residual developer.

[0024] Step six, use a nitrogen gun to blow dry the film. After removing all the moisture, bake the film on a hot plate at 1000°C for 60 seconds. Then, use MEMS lithography technology to etch away the Au film and Cr film outside the photoresist and photoresist protection pattern on the film. The retained Au film and Cr film are the required electrode patterns, and a lithium niobate piezoelectric substrate 3 with a forked electrode 1 structure is obtained. The production of the forked electrode 1 is now completed.

[0025] The method for making the microfluidic channel 2 includes the following contents:

[0026] Step 1: Cover the entire high-resistance silicon surface with photoresist, then place the entire high-resistance silicon with photoresist into a spin coater, and run it in two stages of low speed and high speed in the spin coater, wherein the low speed stage is 1000 rpm for 6 seconds, and the high speed stage is 2500 rpm for 25 seconds.

[0027] Get the negatives;

[0028] Step 2: Place the coated film on a hot plate for baking. Set the hot plate temperature to 1000

[0029] ℃, baking time is 10 minutes;

[0030] Step 3: Use an exposure agent to expose the heated film, then use KMPPD238-II positive photoresist developer to develop the film, then use deionized water to rinse the film twice, each time for 20 seconds to remove the residual developer, and then use a nitrogen gun to blow dry the film;

[0031] Step 4, post-baking the film on a hot plate, the post-baking temperature is set to 100° C., the baking time is 20 minutes, and then a portion of the photoresist on the film is etched away by MEMS photolithography technology, leaving a microchannel pattern on the film, and obtaining a microchannel 2 photolithography mask;

[0032] Step 5: Place the etched film in a glass jar, use acetone ultrasound for 15 minutes to remove the residual positive photoresist, then use ethanol and deionized water to clean the film, and finally use a nitrogen gun to blow dry the film;

[0033] Step 6, placing the negative film in a plastic culture dish, and then placing the plastic culture dish in a fume hood, taking a silanization reagent and dropping it on the blank area on the surface of the plastic culture dish, performing a surface silanization operation on it after natural evaporation, covering the plastic culture dish cover, sealing the plastic culture dish with a sealing film, and reacting in a fume hood overnight; after overnight, opening the sealing film, opening the plastic culture dish cover, leaving it open for 24 hours, taking the plastic culture dish out of the fume hood, and obtaining a microfluidic channel 2 mold template on the plastic culture dish;

[0034] Step 7, the PDMS prepolymer solution and the curing agent are fully mixed and stirred at a mass ratio of 10:1, and then the mixed solution is placed in a vacuum drying oven for vacuum treatment for about 30 minutes to remove bubbles, thereby obtaining a PDMS mixed solution;

[0035] Step 8: Place the microfluidic channel 2 mold template into an open aluminum foil box, pour the PDMS mixed solution prepared in step 7 into the aluminum foil box until the liquid level is 23 mm above the microfluidic channel 2 mold template, and then place the aluminum foil box in a vacuum drying oven for 20 minutes to remove surface bubbles;

[0036] Step nine, transfer the aluminum foil box to a hot plate for heating and curing. The hot plate temperature is set to 85°C and the heating time is 35-45 minutes. After curing is completed, tear off the aluminum foil box to obtain the cured PDMS.

[0037] Step ten, cut the solidified PDMS, take out the preliminary finished product of the microfluidic channel 2, remove excess debris and put it into a glass jar, use ethanol to ultrasonically treat it for 10 minutes, take it out and put it into an oven, dry it at 500°C, and complete the production of the microfluidic channel 2.

[0038] Beneficial effects of the present invention:

[0039] The present invention designs an efficient cell lysis device by optimizing the surface acoustic wave propagation path, microfluidic structure and material selection, thereby achieving rapid and efficient lysis of different types of cells, improving the efficiency of cell disruption, and allowing more cells to be lysed and release internal molecules.

[0040] The present invention uses surface acoustic waves with good mechanical properties and stable transmission characteristics to regulate oscillation force and fluid control, thereby improving the purity and stability of cell lysis and reducing impurities and contamination of samples. Compared with traditional methods, the chip is easy to make, low cost, and low energy consumption. It can perform fast, efficient, and easy-to-control lysis treatment on trace cell samples, and has great value in the field of lysis analysis and biochemical detection of small amounts of cell samples.

[0041] The present invention combines the acoustic capture technology with the microfluidic chip to realize the integrated operation of cell lysis and product collection, and can realize the simultaneous processing of multiple cells, thereby increasing the throughput of sample processing, shortening the experimental time, and improving the operating efficiency and the degree of automation of sample processing.

[0042] The present invention connects the interdigital electrodes to a 24V DC power supply, with an input power of 1.5W, which generates a frequency of about 30.5MHz for the propagation of surface acoustic waves, and can generate a signal at a cell density of 2×10 in about 20 seconds. 5A cell lysis efficiency of 92% was achieved in a 5-microliter droplet of 400 μg / ml, thereby achieving rapid and efficient cell lysis in microliter-level cell liquids. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings used in the description of the embodiments of the present invention. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the contents of the embodiments of the present invention and these drawings without creative work.

[0044] Figure 1 The schematic diagram of the structure of the cell lysis microfluidic biochip based on the surface acoustic wave principle of the present invention.

[0045] Figure 2 Schematic diagram of the structural details of the interdigitated electrode of the present invention.

[0046] Figure 3 Schematic diagram of the microfluidic channel structure of the present invention.

[0047] Figure 4 Schematic diagram of the structural details of the microfluidic lysis chamber and micro cylindrical baffle of the present invention.

[0048] Figure 5 It is a schematic diagram of the simulation results of the vibration mode of the interdigitated electrode of the present invention at a specific characteristic frequency.

[0049] Figure 6 Schematic diagram of simulation results of the motion trajectory of magnetic particles in the microfluidic lysis chamber of the present invention.

[0050] Figure 7 This is a diagram of the cell lysis process of the cell lysis microfluidic biochip based on the surface acoustic wave principle of the present invention. DETAILED DESCRIPTION

[0051] The present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. It is to be understood that the specific embodiments described herein are only used to explain the present invention, rather than to limit the present invention. It should also be noted that, for ease of description, only parts related to the present invention, rather than all structures, are shown in the accompanying drawings.

[0052] Example 1

[0053] A cell lysis microfluidic biochip based on the surface acoustic wave principle, such as Figure 1-4 As shown, it includes an interdigital electrode 1, a microchannel 2 and a lithium niobate piezoelectric substrate 3 that are bonded and packaged together, wherein the microchannel 2 and the interdigital electrode 1 are fixed on the lithium niobate piezoelectric substrate 3 in sequence from left to right;

[0054] The interdigitated electrode 1 includes a plurality of electrode pairs and two front and rear bus bars 16, wherein a plurality of electrode pairs are arranged between the front and rear bus bars 16, each electrode pair includes two left and right electrode fingers 11 and a suspended electrode structure 15 located between the electrode fingers 11, the bottom of the electrode fingers 11 is fixed on the lithium niobate piezoelectric substrate 3, and the rear end of the left electrode finger 11 is fixed on the rear bus bar 16, and the front end of the right electrode finger 11 is fixed on the front bus bar 16, the suspended electrode structure 15 includes a gate-type finger 13 and a suspended finger 12, wherein two evenly distributed suspended fingers 12 are arranged inside the gate-type finger 13, and the bottoms of the gate-type finger 13 and the suspended finger 12 are both fixed on the lithium niobate piezoelectric substrate 3 and are not connected to the bus bar 16;

[0055] The microfluidic channel 2 includes a microfluidic plate, magnetic particles 26 and a microcolumn baffle 27, wherein the front, middle and rear ends of the microfluidic plate are respectively provided with an inlet 21, a lysis chamber 23 and an outlet 25, wherein the inlet 21 is connected with the lysis chamber 23 through an inlet channel 22, and the lysis chamber 23 and the outlet 25 are connected through an outlet channel 24, and a microcolumn baffle 27 is provided at one end of the lysis chamber 23 close to the outlet channel 24, and a plurality of magnetic particles 26 are placed in the lysis chamber 23.

[0056] The micro-column baffle 27 includes a row of multiple micro-columns arranged in an arc shape, and is placed at one end of the lysis chamber 23 close to the outlet flow channel 24 and fixed in the lysis chamber 23 .

[0057] The inlet 21 and the outlet 25 are through holes, and the inlet channel 22, the lysis chamber 23 and the outlet channel 24 are all grooves dug out on the microchannel plate, that is, grooves with an open bottom and a closed top.

[0058] The bus bar 16 includes a long bar and a short bar, wherein the short bar is fixed outside the right end of the long bar.

[0059] A method for realizing cell lysis by using a cell lysis microfluidic biochip based on the surface acoustic wave principle and combining surface acoustic wave and microfluidic technology, such as Figure 7 As shown, the following steps are included:

[0060] Step S1-1: Connect a power source to one end of the short strip of the two bus bars 16 on the interdigital electrode 1, i.e., the input end, and simultaneously introduce the liquid to be lysed into the microchannel 2 through the inlet 21;

[0061] Step S1-2: After the interdigital electrodes 1 are energized, surface acoustic waves are excited on the lithium niobate piezoelectric substrate 3 and propagate toward the microchannel 2;

[0062] Step S1-3: The sound waves drive the cells in the liquid to be lysed in the microfluidic channel 2 to collide rapidly with the magnetic particles 26, thereby achieving cell lysis; wherein the liquid to be lysed flows into the lysis chamber 23 through the inlet 21 and the inlet channel 22. The process of introducing the liquid is continuous, and the unlysed cells cannot pass through the microcolumn baffle 27 composed of micro-cylinders. The contents after cell lysis will continue to flow out at the outlet 25 along with the liquid through the outlet channel 24, thereby achieving continuous cell lysis and timely collection of lysis products.

[0063] The method for manufacturing the interdigital electrode 1 includes the following contents:

[0064] Step 1: The interdigitated electrode 1 is manufactured by a thin film process, with a lithium niobate piezoelectric substrate 3 as a base film, a Cr film is deposited on the base film by a thermal evaporation device, and then an Au film is deposited on the Cr film; in the step 1, the Cr film is used as an adhesion layer to improve the adhesion between the Au layer and the substrate, and its thickness is generally 5nm; the Au film is used as the electrode surface layer, and the thickness of the vapor deposition is generally between 80-120nm.

[0065] Step 2: Cover the entire surface of the film after the evaporation in step 1 with a photoresist (the thickness of the photoresist is 800 nm) through a spin coating process, and then place the film with the photoresist into a spin coater, and run the spin coater at two speeds, low speed and high speed, respectively, wherein the low speed stage is 1000 rpm for 5 seconds, and the high speed stage is 5500 rpm for 30 seconds, to obtain a film after the coating is evenly coated;

[0066] Step 3: Place the coated film on a hot plate and bake it. The hot plate temperature is set to 110°C and the baking time is 2 minutes.

[0067] Step 4: Use 123 mL of exposure agent to expose the heated film. After the exposure is completed, place the film on a hot plate for baking. The hot plate temperature is set to 100° C. and the baking time is 80 seconds to fix the connection between the photoresist and the film.

[0068] Step 5: Take 100 mL of KMPPD238-II positive photoresist developer and 40 mL of deionized water and mix them evenly. Place the baked film in the mixed solution and develop for 50 seconds. Then, rinse the film twice with deionized water, each time for 20 seconds to remove the residual developer.

[0069] Step six, use a nitrogen gun to slowly blow dry the film along the surface. After removing all the moisture, dry the film on a hot plate at 1000°C for 60 seconds. Then, use MEMS lithography technology to etch away the Au film and Cr film outside the photoresist and photoresist protection pattern on the film. The retained Au film and Cr film are the required electrode patterns, and a lithium niobate piezoelectric substrate 3 with a forked electrode 1 structure is obtained. The production of the forked electrode 1 is now completed.

[0070] The method for making the microfluidic channel 2 includes the following contents:

[0071] Step 1, covering the entire high-resistance silicon surface with a photoresist (the thickness of the photoresist is about 3 μm), and then placing the entire high-resistance silicon with the photoresist in a spin coater, and operating the spin coater in two stages of low speed and high speed, wherein the low speed stage is 1000 rpm for 6 seconds, and the high speed stage is 2500 rpm for 25 seconds, to obtain a negative film;

[0072] Step 2: Place the coated film on a hot plate for baking. Set the hot plate temperature to 1000

[0073] ℃, baking time is 10 minutes;

[0074] Step 3: Use 123 mL of exposure agent to expose the heated film, then use KMPPD238-II positive photolithography developer to develop the film, take 100 mL of the developer into a glass jar, develop for 50 seconds, then rinse the film with deionized water for two rounds, each for 20 seconds to remove the residual developer, and then use a nitrogen gun to blow dry the film;

[0075] Step 4: To ensure the stability of the pattern in the subsequent dry etching, the film needs to be post-baked on a hot plate. The post-baking temperature is set to 100°C and the baking time is 20 minutes. Then, part of the photoresist on the film is etched away by MEMS lithography technology, leaving a microchannel pattern on the film.

[0076] Obtaining a microfluidic channel 2 photolithography mask;

[0077] Step 5: After the microfluidic channel 2 photolithography mask is photolithographically processed, the etched film is placed in a glass tank, and acetone is ultrasonically treated for 15 minutes to remove the residual positive photoresist. Then, ethanol and deionized water are used to clean the film surface until a clean surface is obtained. Finally, a nitrogen gun is used to blow dry the film.

[0078] Step 6: Place the film in a plastic culture dish with a diameter of 10 cm, and then place the plastic culture dish in a fume hood. Carefully take a silanization reagent (trichloro(1H,1H,2H,2H-perfluorooctyl)silane, Sigma) and drip it on the blank area on the surface of the plastic culture dish. After natural evaporation, perform a surface silanization operation on it. Cover the plastic culture dish with a lid, carefully seal the plastic culture dish with a sealing film, and react overnight in a fume hood.

[0079] After overnight, the sealing film was opened, the lid of the plastic culture dish was opened, and the dish was left open for 24 hours until the gas was completely dispersed, and the plastic culture dish was taken out of the fume hood to obtain the microfluidic channel 2 mold template on the plastic culture dish;

[0080] Step 7, the PDMS prepolymer solution and the curing agent are fully mixed and stirred at a mass ratio of 10:1, and then the mixed solution is placed in a vacuum drying oven for vacuum treatment for about 30 minutes to remove bubbles, thereby obtaining a PDMS mixed solution;

[0081] Step 8: Fold the aluminum foil into an open aluminum foil box (the size of the bottom of the box is exactly the same as the size of the template), put the microfluidic channel 2 mold template into the open aluminum foil box, pour the PDMS mixed liquid prepared in step 7 into the aluminum foil box until the liquid level is 23 mm above the microfluidic channel 2 mold template, and then put the aluminum foil box into a vacuum drying oven for vacuum treatment for 20 minutes to completely remove surface bubbles;

[0082] Step nine, transfer the aluminum foil box to a hot plate for heating and curing. The hot plate temperature is set to 85°C and the heating time is 35-45 minutes. After curing is completed, tear off the aluminum foil box to obtain the cured PDMS.

[0083] Step ten, cut the solidified PDMS, take out the preliminary finished product of the microfluidic channel 2, remove excess debris and put it into a glass jar, use ethanol to ultrasonically treat it for 10 minutes, take it out and put it into an oven, dry it at 500°C, and complete the production of the microfluidic channel 2.

[0084] Example 2

[0085] The present invention provides a method for rapidly and efficiently lysing cells in microliter-level liquids. The present invention applies SAW acoustic imaging technology and microfluidics technology to the field of biomedical materials to reduce the sample demand in cell detection, speed up the detection speed, and avoid potential harm to operators during the process, thereby achieving rapid and efficient cell lysis with low cost and low energy consumption.

[0086] In this embodiment, a first aspect provides a cell lysis microfluidic biochip based on the surface acoustic wave principle.

[0087] Specifically, Figure 1As shown, the cell lysis microfluidic biochip based on the surface acoustic wave principle includes interdigital electrodes 1, microchannels 2 and a lithium niobate piezoelectric substrate 3.

[0088] In this embodiment, the material of the interdigital electrodes 1 is gold, a high-frequency design is used, and a suspended electrode is used for anti-reflection design to reduce the appearance of clutter to achieve more accurate surface acoustic wave excitation and droplet driving.

[0089] Specifically, Figure 2 As shown, the details of the interdigital electrode 1 are the bus bar 16 and the interdigital part, and its key parameters are that the width of the electrode finger 11 is 10 μm, the interval between the electrode finger 11 and the side bar of the gate-type finger 13, the interval between each two suspended fingers 12, and the interval between the suspended finger 12 and the side bar of the gate-type finger 13 are all 10 μm; the length of the gate-type finger 13 and the suspended finger 12 (the width of the intersection of the two sides of the interdigital electrode 1) is 9.2 mm; the thickness of the electrode finger 11, the suspended electrode structure 15 and the bus bar 16 are all 0.1 μm, and the number of electrode pairs is 80 pairs. This electrode adopts the suspended electrode structure 15 for anti-reflection design. This electrode construction method can greatly reduce the appearance of clutter to achieve more accurate surface acoustic wave excitation and droplet driving. A layer of chromium film is also covered between the interdigital electrode 1 and the lithium niobate piezoelectric substrate 3 to enhance adhesion so that the interdigital electrode 1 is fixed on the lithium niobate piezoelectric substrate 3.

[0090] One end of the electrode finger 11 is directly connected to the bus bar 16, the gate-shaped finger 13 and the two suspended fingers 12 in the middle are independently suspended and not connected to the bus bar 16. The electrode period is the overall length covered by a pair of electrodes, and its size determines the effective width of the surface acoustic wave, so its value will affect the specific sound field structure.

[0091] Specifically, Figure 3 As shown, the microfluidic channel 2 is formed by photolithography of a PDMS thin layer, and is mainly composed of four parts, namely, an inlet 21, an inlet channel 22, a cracking chamber 23, an outlet channel 24 and an outlet 25. The inlet 21 and the outlet 25 are both designed with cylindrical through-hole structures, and a micro hose can be inserted to facilitate the liquid to be cracked to enter the microfluidic channel 2 and the cracked liquid to leave the microfluidic channel 2. The material used in the microfluidic channel 2 is PDMS (polydimethylsiloxane), which is transparent, colorless and tasteless, has good physical and chemical stability, and is convenient for experimental observation. The inlet channel 22, the cracking chamber 23, and the outlet channel 24 are arranged inside the microfluidic channel 2 structure, and are cavities dug out from the bottom surface of the microfluidic channel 2, and are not through from top to bottom, which is equivalent to the microfluidic channel 2 being covered on the lithium niobate piezoelectric substrate.

[0092] The piezoelectric substrate is composed of lithium niobate (128° YX-LiNbO3).

[0093] In this embodiment, a micro cylindrical baffle 27 is provided at the connection between the lysis chamber 23 and the outlet flow channel 24. The micro cylindrical baffle 27 includes a plurality of cylinders fixed to the inner edge of the lysis chamber 23.

[0094] Specifically, Figure 4 As shown, cells are lysed in the lysis chamber 23 by colliding with magnetic particles, and a row of arc-shaped micro-cylindrical baffles 27 are arranged at the end of the lysis chamber 23 to prevent the magnetic particles 26 from flowing out with the lysis solution, thereby confining the magnetic particles 26 and unlysed cells in the lysis chamber without affecting the outflow of the cell lysis solution, and at the same time, the chip cell lysis effect can be improved. The magnetic particles 26 are suspended in the liquid of the lysis chamber 23 of the microfluidic channel 2.

[0095] In this embodiment, a method for lysing cells using surface acoustic waves and microfluidics technology is proposed in the second aspect, including the generation and control of surface acoustic waves and how surface acoustic waves control cell lysis.

[0096] The interdigital electrodes 1 deform the piezoelectric material by applying an electric field on the lithium niobate piezoelectric substrate 3, thereby generating surface acoustic waves on the surface of the substrate material. The number of electrode pairs N of the interdigital electrodes 1 determines the intensity of the surface acoustic wave, the electrode aperture W determines the width of the surface acoustic wave action area, and the electrode period M affects the excitation frequency of the surface acoustic wave.

[0097] The surface acoustic wave is used to generate force on the fluid in the microchannel 2 when propagating in the microchannel 2, thereby achieving control and operation of the fluid.

[0098] When the surface acoustic wave propagates in the microchannel 2, it generates a force on the fluid therein to achieve control and operation of the fluid. During the cell lysis process, the surface acoustic wave couples into the acoustic pressure field of the liquid at the Rayleigh angle and generates pressure intensity distribution nodes, causing the liquid to produce an acoustic microfluidic effect, thereby achieving accelerated movement of particles and cells in the liquid and achieving collision cell lysis.

[0099] like Figure 7 As shown, the specific steps include:

[0100] Step S1-1: Connect the relevant instruments and power supply to the wider end of the two bus bars 16 on the interdigital electrode 1, i.e., the input end; that is, connect the bus bar 16 of the interdigital electrode 1 on the chip to the Agilent 33250A arbitrary waveform signal generator, and select the ZHL-20+ signal amplifier, equipped with a 24V constant voltage power supply for driving;

[0101] In this embodiment, a coaxial RF signal connection line is used to connect the output end of the signal generator to the input end of the power amplifier, a DC power supply is correctly connected to the voltage interface of the power amplifier, and the DC power supply is turned on to adjust the output voltage to 24V and the output current to 3A; after observing that the fan of the power amplifier is working normally, a coaxial RF signal connection line is used to connect the output end of the power amplifier to the input end of the surface acoustic wave cell lysis device, that is, the input end of the interdigital electrode 1, to complete the connection between the interdigital electrode 1-1 and the test system;

[0102] Step S1-2: After the interdigital electrodes 1 are energized, surface acoustic waves are excited on the lithium niobate piezoelectric substrate 3 and propagate toward the microchannel 2;

[0103] In this embodiment, after the interdigital electrodes 1 are energized, the lithium niobate piezoelectric substrate 3 underneath is deformed due to the piezoelectric effect, and then a surface acoustic wave with a wavelength of 0.04 mm is generated, which propagates toward the microchannel 2 at a speed of about 3992 m / s.

[0104] Insert a plastic hose of suitable size into the inlet 21 of the microfluidic channel 2, and pass the liquid to be lysed containing cells into the microfluidic channel 2 through the plastic hose, and then flow into the lysis chamber 23 through the inlet 21 and the inlet channel 22 in sequence;

[0105] Step S1-3: The sound waves drive the cells in the liquid sample in the microfluidic channel 2 to collide quickly with the magnetic particles 26, thereby achieving cell lysis; the liquid to be lysed flows into the lysis chamber 23 through the inlet 21 and the inlet channel 22. The process of introducing the liquid is continuous, and the unlysed cells cannot pass through the micro cylindrical baffle 27. The contents after cell lysis will continue to flow out at the outlet 25 along with the liquid through the outlet channel 24. The lysis time is about 20s.

[0106] In this embodiment, when the surface acoustic wave is transmitted to the microfluidic channel 2, the liquid in the lysis chamber 23 is driven to move, and the cells and magnetic particles 26 in the liquid move with the liquid, and collide at high speed during the process, thereby causing cell lysis and spillage of cell contents. A plastic hose of suitable size is inserted into the outlet 25 of the microfluidic channel 2, and the liquid is waited to continue to flow out of the plastic hose at the outlet 25;

[0107] On the other hand, the sample liquid continuously flows in and out of the microfluidic channel 2, so as to achieve continuous lysis of cells and timely collection of lysis products.

[0108] The above method of lysing cells using surface acoustic waves and microfluidics technology has the following beneficial effects:

[0109] This embodiment is based on the surface acoustic wave principle and applies SAW acoustic pickup technology and microfluidic technology to the field of biomedical materials. It can realize the cell lysis function at the micro-nano level at the cellular level. Compared with traditional cell lysis detection technology, this method overcomes the problems of large sample volume requirement, poor lysis effect, potential harm to operators, etc., and realizes rapid, efficient and controllable lysis of cells in microliter-level liquids, which has great application significance in the processing of biological samples.

[0110] In this embodiment, in a third aspect, a simulation analysis method for a cell lysis microfluidic biochip based on the surface acoustic wave principle is proposed.

[0111] Specifically, Figure 5 As shown, a model is established for one of the fingers of the interdigital electrode 1 in professional simulation software, gold is selected as the material, electrostatics and solid mechanics physical fields are selected and relevant parameters are set, and a simulation analysis of the piezoelectric effect is performed. Finally, the simulation result that the interdigital electrode 1 produces the most obvious vibration mode at the characteristic frequency of 19.432Mhz is obtained.

[0112] Specifically, Figure 6 As shown, a model of the microfluidic channel 2 is established in a professional simulation software, PDMS is selected as the material, laminar flow and solid mechanics physical fields are selected and relevant parameters are set, and a simulation analysis of fluid-solid coupling is performed. Finally, the simulation results of the movement trajectory of the magnetic particles 26 in the lysis chamber 23 are obtained under the condition of continuously introducing the liquid to be lysed into the microfluidic channel 2.

[0113] By simulating the movement trajectory of the interdigital electrodes 1 and the magnetic particles 26 in the microchannel 2, this embodiment can obtain a clearer operating status of each component of the chip when it is working, which is helpful for structural optimization design and related performance testing of the chip.

[0114] In this embodiment, a fourth aspect provides a method for preparing a cell lysis microfluidic biochip based on the surface acoustic wave principle, comprising the following steps:

[0115] Step S21: drawing a photolithography mask for the interdigital electrodes 1 and the microfluidic channels 2 according to a pre-designed design;

[0116] Step S22: using micro-nano processing technology based on photolithography to process the interdigitated electrodes 1 and the microchannels 2;

[0117] Specifically, for the interdigital electrode 1, there is no strict requirement for the substrate but the graphic accuracy requirement is high, so the stripping method is used for processing. The specific steps of the method are: coating, photolithography, gold evaporation, stripping, and thin film technology is used to evaporate and deposit Cr / Au thin film through a thermal evaporation deposition apparatus to complete it. The Cr layer is used as an adhesion layer to improve the adhesion between the Au layer and the substrate, and its thickness is generally 5nm. The Au thin film is used as the electrode surface layer, and the thickness of the evaporation is generally between 80-120nm, so that the device has good working performance.

[0118] Specifically, for the microfluidic channel 2, first, a photolithography mask containing a pre-designed microfluidic pattern needs to be made, and the mask is usually made of quartz or glass as a substrate. After the mask is made, a photoresist is coated on the silicon wafer substrate, and the mask is aligned with the substrate coated with the photoresist and then exposed; after the exposure is completed, the substrate is immersed in a developer to remove excess photoresist and retain the pattern of the microfluidic channel. Since its graphic structure is relatively simple but needs to be processed evenly and reused, it is processed by a dry etching method. The specific steps of this method are: gold evaporation, glue uniformity, photolithography, etching, and the etching depth is 60μm. After the etching is completed, the photoresist needs to be removed and cleaned with ethanol and deionized water, and then blown dry with a nitrogen gun, and then the surface is silanized to complete the processing of the microfluidic channel 2 mold template. Finally, the PDMS prepolymer and curing agent are injected into the mold template, and the PDMS is removed from the template after heating and curing. The target PDMS microfluidic channel structure is prepared by subsequent treatments such as removing excess materials and punching.

[0119] Step S23: Bond and package the processed interdigital electrodes 1 and the microchannels 2 to build a surface acoustic wave excitation and testing system.

[0120] Specifically, since the PDMS microfluidic channel 2 is an open structure, in order to close the channel, the two need to be bonded and packaged to form a closed microfluidic channel with the lithium niobate piezoelectric substrate 3. In order to make the overall device firm and not prone to peeling, a bonding method after surface treatment is adopted, and the bonding surface is treated with ultraviolet light or plasma to make active groups appear on the surface, and the surface of the microfluidic channel 2 and the lithium niobate piezoelectric substrate 3 are closely attached to form a stable chemical bond, so that the overall structure of the device is firm and stable.

[0121] The present invention applies a cell lysis microfluidic biochip to the field of biomedical materials, solving the problems of slow detection speed, large sample demand, and potential harm to operators in current cell detection.

[0122] Compared with traditional methods, the present invention uses SAW acoustic imaging technology to perform cell lysis, which reduces costs and energy consumption and achieves rapid and efficient cell lysis in trace amounts of liquid.

[0123] The present invention utilizes microfluidic technology to achieve high controllability of the cell lysis process, making it easy to collect, analyze and detect cell lysis products.

[0124] The preferred embodiments of the present invention are described in detail above in conjunction with the accompanying drawings. However, the protection scope of the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention. These simple variations all belong to the protection scope of the present invention.

[0125] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0126] In addition, various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims

1. A cell lysis microfluidic biochip based on the surface acoustic wave principle, characterized in that: It comprises an interdigital electrode (1), a microfluidic channel (2) and a lithium niobate piezoelectric substrate (3), wherein the microfluidic channel (2) and the interdigital electrode (1) are fixed on the lithium niobate piezoelectric substrate (3) in sequence from left to right; The interdigitated electrode (1) comprises a plurality of electrode pairs and a bus bar (16), wherein a plurality of electrode pairs are arranged between the bus bars (16), the electrode pairs comprise two left and right electrode fingers (11) and a suspended electrode structure (15) located between the electrode fingers (11), the bottom of the electrode fingers (11) is fixed on a lithium niobate piezoelectric substrate (3), and the rear end of the left electrode finger (11) is fixed on the rear end bus bar (16), and the front end of the right electrode finger (11) is fixed on the front end bus bar (16), and the suspended electrode structure (15) comprises a gate-type finger (13) and a suspended finger (12), wherein the gate-type finger (13) is provided with uniformly distributed suspended fingers (12), and the bottoms of the gate-type finger (13) and the suspended finger (12) are both fixed on the lithium niobate piezoelectric substrate (3); The microfluidic channel (2) comprises a microfluidic channel plate, magnetic particles (26) and a microcolumn baffle (27), wherein the front, middle and rear ends of the microfluidic channel plate are respectively provided with an inlet (21), a lysis chamber (23) and an outlet (25), wherein the inlet (21) is connected to the lysis chamber (23) through an inlet channel (22), the lysis chamber (23) and the outlet (25) are connected through an outlet channel (24), a microcolumn baffle (27) is provided at one end of the lysis chamber (23) close to the outlet channel (24), and a plurality of magnetic particles (26) are placed in the lysis chamber (23).

2. A cell lysis microfluidic biochip based on the surface acoustic wave principle according to claim 1, characterized in that: The micro-column baffle (27) comprises a row of a plurality of micro-columns arranged in an arc shape, and is placed at one end of the lysis chamber (23) close to the outlet flow channel (24) and fixed in the lysis chamber (23).

3. The cell lysis microfluidic biochip based on the surface acoustic wave principle according to claim 1, characterized in that: The inlet (21) and the outlet (25) are both through holes.

4. The cell lysis microfluidic biochip based on the surface acoustic wave principle according to claim 1, characterized in that: The bus bar (16) comprises a long bar and a short bar, wherein the short bar is fixed on the outside of the right end of the long bar.

5. A method for realizing cell lysis by using surface acoustic wave and microfluidic technology by using the cell lysis microfluidic biochip based on the surface acoustic wave principle as described in any one of claims 1 to 4, characterized in that: The steps include: Step S1-1: connecting a power source to one end of the short strip of two bus bars (16) on the interdigitated electrode (1), i.e., the input end, and simultaneously introducing the liquid to be lysed into the microchannel (2) through the inlet (21); Step S1-2: After the interdigital electrodes (1) are energized, surface acoustic waves are generated on the lithium niobate piezoelectric substrate (3) and propagate toward the microchannel (2); Step S1-3: The sound waves drive the cells in the liquid to be lysed in the microchannel (2) to collide rapidly with the magnetic particles (26), thereby achieving cell lysis; wherein the liquid to be lysed flows into the lysis chamber (23) through the inlet (21) and the inlet channel (22), and the process of introducing the liquid is continuous. Unlysed cells cannot pass through the microcolumn baffle (27) composed of micro-cylinders, and the contents after cell lysis will continue to flow out at the outlet (25) along with the liquid through the outlet channel (24), thereby achieving continuous cell lysis and timely collection of lysis products.

6. A method for manufacturing the interdigital electrode (1) of the cell lysis microfluidic biochip based on the surface acoustic wave principle as claimed in any one of claims 1 to 4, characterized in that: It includes the following: Step 1: Using a lithium niobate piezoelectric substrate (3) as a base film, depositing a Cr film on the base film by a thermal evaporation deposition apparatus, and then depositing an Au film on the Cr film; Step 2, covering the entire surface of the film after the evaporation in step 1 with photoresist, and then placing the film with the photoresist in a spin coater, and running the spin coater at two speeds, low speed and high speed, respectively, wherein the low speed stage is 1000 rpm for 5 seconds, and the high speed stage is 5500 rpm for 30 seconds, to obtain a film after photoresist is evenly coated; Step 3: Place the coated film on a hot plate and bake it. The hot plate temperature is set to 110°C and the baking time is 2 minutes. Step 4, using an exposure agent to expose the heated film, and after the exposure is completed, the film is placed on a hot plate for baking, the hot plate temperature is set to 100° C., and the baking time is 80 seconds to fix the connection between the photoresist and the film; Step 5: Take 100 mL of KMPPD238-II positive photoresist developer and 40 mL of deionized water and mix them evenly. Place the baked film in the mixed solution and develop for 50 seconds. Then, rinse the film twice with deionized water, each time for 20 seconds to remove the residual developer. Step six, use a nitrogen gun to blow dry the film, remove all moisture, and then bake the film on a hot plate at 1000°C for 60 seconds. Then, use MEMS photolithography technology to etch away the Au film and Cr film outside the photoresist and photoresist protection pattern on the film. The retained Au film and Cr film are the required electrode patterns, and a lithium niobate piezoelectric substrate (3) with a forked electrode (1) structure is obtained. At this point, the production of the forked electrode (1) is completed.

7. A method for manufacturing the microchannel (2) of the cell lysis microfluidic biochip based on the surface acoustic wave principle as claimed in any one of claims 1 to 4, characterized in that: It includes the following: Step 1: Cover the entire high-resistance silicon surface with photoresist, then place the entire high-resistance silicon with photoresist into a spin coater, and run the spin coater in two stages of low speed and high speed, wherein the low speed stage is 1000 rpm for 6 seconds, and the high speed stage is 2500 rpm for 25 seconds, to obtain a negative film; Step 2: Place the coated film on a hot plate for baking. The hot plate temperature is set to 1000°C and the baking time is 10 minutes. Step 3: Use an exposure agent to expose the heated film, then use KMPPD238-II positive photoresist developer to develop the film, then use deionized water to rinse the film twice, each time for 20 seconds to remove the residual developer, and then use a nitrogen gun to blow dry the film; Step 4, post-baking the film on a hot plate, the post-baking temperature is set to 100° C., the baking time is 20 minutes, and then a portion of the photoresist on the film is etched away by MEMS photolithography technology, leaving a microfluidic pattern on the film, to obtain a microfluidic (2) photolithography mask; Step 5: Place the etched film in a glass jar, use acetone ultrasound for 15 minutes to remove the residual positive photoresist, then use ethanol and deionized water to clean the film, and finally use a nitrogen gun to blow dry the film; Step 6, placing the film in a plastic culture dish, and then placing the plastic culture dish in a fume hood, taking a silanization reagent and dripping it on the blank area on the surface of the plastic culture dish, performing a surface silanization operation on it after natural evaporation, covering the plastic culture dish with a lid, and sealing the plastic culture dish with a sealing film, and reacting in a fume hood overnight; after overnight, open the sealing film, open the plastic culture dish lid, and leave it open for 24 hours, and then take the plastic culture dish out of the fume hood to obtain a microfluidic channel (2) mold template on the plastic culture dish; Step 7, the PDMS prepolymer solution and the curing agent are fully mixed and stirred at a mass ratio of 10:1, and then the mixed solution is placed in a vacuum drying oven for vacuum treatment for about 30 minutes to remove bubbles, thereby obtaining a PDMS mixed solution; Step 8: Place the microfluidic channel (2) mold template into an open aluminum foil box, pour the PDMS mixed solution prepared in step 7 into the aluminum foil box until the liquid level is 23 mm above the microfluidic channel (2) mold template, and then place the aluminum foil box in a vacuum drying oven for 20 minutes to remove surface bubbles; Step nine, transfer the aluminum foil box to a hot plate for heating and curing. The hot plate temperature is set to 85°C and the heating time is 35-45 minutes. After curing is completed, tear off the aluminum foil box to obtain the cured PDMS. Step 10, cut the solidified PDMS, take out the preliminary finished product of the microfluidic channel (2), remove excess debris and put it into a glass jar, use ethanol to ultrasonically treat it for 10 minutes, take it out and put it into an oven, dry it at 500°C, and complete the production of the microfluidic channel (2).

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