WO2022120769A1

WO2022120769A1 - High-throughput lysis system based on resonant micro-bubble array

Info

Publication number: WO2022120769A1
Application number: PCT/CN2020/135486
Authority: WO
Inventors: 郑海荣; 刘秀芳; 孟龙; 徐礼胜; 荣宁; 牛丽丽
Original assignee: 深圳先进技术研究院
Priority date: 2020-12-10
Filing date: 2020-12-10
Publication date: 2022-06-16

Abstract

Provided is a high-throughput lysis system based on a resonant micro-bubble array, and specifically disclosed is a lysis apparatus. The apparatus comprises: 1) a cavity for forming a micro-bubble array, the cavity being provided with a fluid cavity channel, wherein two sides of the fluid cavity channel are provided with micro-structure cavities that are in communication with the cavity channel and protrude outward, and the microstructure cavities can capture and keep micro-bubbles; 2) a substrate, the substrate and the cavity being bonded and together forming, between the substrate and the fluid cavity channel of the cavity, a fluid channel capable of allowing a fluid to pass therethrough; and 3) a body wave generation device, the body wave generation device being capable of generating body waves and transmitting same into the cavity where the micro-bubble array is formed, and the body waves resonating with the micro-bubbles. The apparatus structure of the present invention is simple, and high-throughput lysis of cells, bacteria, viruses or tissue can be achieved.

Description

A high-throughput lysis system based on resonant microbubble arrays

technical field

The invention belongs to the field of molecular biology, and in particular relates to a system for rapidly lysing cells or viruses based on a resonance microbubble array.

Background technique

In recent years, with the importance of isolation and purification of cellular components in disease diagnosis, gene therapy, cell engineering, and drug screening, cell lysis has attracted widespread attention. Cell lysis is the process of breaking the nuclear envelope to release intracellular material such as DNA, RNA, proteins or organelles. However, developing an efficient and rapid cell lysis technique is crucial for the isolation and purification of intracellular components. At present, common cell lysis techniques are divided into two categories: mechanical lysis and non-mechanical lysis. Although the mechanical lysis method can achieve a high-throughput lysis effect, it is easy to produce a strong thermal effect and is not suitable for extracting intracellular proteins. In contrast, non-mechanical cell lysis techniques are more popular. Biological and chemical non-mechanical lysis techniques can prevent mechanical damage to intracellular components, and are especially suitable for mechanosensitive intracellular components, but have disadvantages such as expensive reagents and complicated operations. As the most promising method in non-mechanical cell lysis technology, physical cell lysis technology is widely used in experiments and practical production. At present, the most commonly used non-mechanical cell lysis technologies are biological lysis technology, chemical lysis technology, temperature lysis technology, osmotic pressure lysis technology, photolysis technology, electrolysis technology and acoustic lysis technology.

Biological lysis technology: Enzymatic lysis technology is a commonly used method. Enzymatic lysis is a biological cell lysis method. There are many types of enzymes, and specific types of cells require specific lysis enzymes. For example, lysozyme is used for bacterial cell lysis, chitin Plasmidase can be used for yeast cell lysis, while pectinase is used for plant cell lysis. The biggest advantage of the enzymatic cleavage method is specificity. However, due to the specificity of the enzyme, its limitations are limited and it is not conducive to wide application. And there is a problem that the cells cannot be completely lysed. ([1] Andrews, B.A.; Asenjo, J.A. Enzymatic lysis and disruption of microbial cells. Trends Biotechnol. 1987, 5, 273–277. [2] Salazar, O.; Asenjo, J.A. Enzymatic lysis of microbial cells. Biotechnol. Lett. 2007, 29, 985–994.)

Chemical lysis technique: disrupts the cell membrane by changing the pH of the chemical lysis solution. Surfactants can also be added to cell lysis buffers to dissolve membrane proteins to disrupt cell membranes and release their contents. Chemical cracking technology can be divided into alkaline cracking method and surfactant cracking method. In alkaline lysis technology, ^OH- ion is the main component of lysis cell membrane. Lysis buffer consisted of sodium hydroxide and sodium dodecyl sulfate (SDS). OH ^– can break the fatty acid-glyceride bond in the cell membrane, which subsequently makes the cell membrane permeable, and SDS dissolves proteins and membranes. The pH range of the lysate is generally controlled to 11.5–12.5. In the surfactant splitting technology, the surfactant can destroy the double lipid layer composed of hydrophobic and hydrophilic molecules in the cell membrane, the main principle is to destroy the lipid-lipid, lipid-protein and protein-protein in the cell membrane interaction force. Although this method works for all cell types, the cell lysis process is very slow, taking approximately 6 to 12 hours. ([3] Stanbury, PF; Whitaker, A. Principles of Fermentation Technology; Pergamon Press: Oxford, UK, 1984. [4] Feliciello, I.; Chinali, GA modified alkaline lysis method for the preparation of highly purified plasmid DNA from Escherichia coli. Anal. Biochem. 1993, 212, 394–401.)

Temperature lysis technology: It is a technology that causes ice crystals to form on the cell membrane by repeated freezing and thawing of cells, so that the cell membrane is decomposed, resulting in cell lysis. At the same time, high temperature can also damage the membrane through the denaturation of cell membrane proteins and lead to the release of intracellular organelles. However, although this lysis technique is not limited to cell types and is easy to implement, it is time-consuming, expensive, and cannot be used to extract temperature-sensitive intracellular components. ([5]Johnson,B.H.;Hecht,M.H.Cells by repeated cycles of freezing and thawing.Biotechnology 1994,12,1357;Zhu,K.[6]Jin,H.;He,Z.;Zhu,Q.;Wang ,B.A continuous method for the large-scale extraction of plasma DNA by modified boiling lysis.Nat.Protoc.2007,1,3088–3093.)

Osmotic lysis technology: When the concentration of extracellular fluid is lower than that of intracellular fluid, the extracellular fluid will enter the cell due to osmosis, which will cause the cell to swell and subsequently rupture. Due to the fragile structure of mammalian cell membranes, this technique is suitable for lysing mammalian cells and can be used to extract sensitive components within cells, but this technique is not suitable for all types of cells, so it is largely limited The application of this cracking technology. ([7]Fonseca, L.P.; Cabral, J.Penicillin acylase release from Escherichia coli cells by mechanical cell disruption and permeabilization.J.Chem.Technol.Biotechnol.2002,77,159–167.[8]Chen,Y.-C .;Chen,L.-A.;Chen,S.-J.;Chang,M.-C.;Chen,T.-L.A modified osmotic shock for periplasmic release of a recombinant creatinase from Escherichia coli.Biochem.Eng. J. 2004, 19, 211–215.)

Photolysis technology: It is a kind of laser targeting to control cells. After laser irradiation, the focused laser pulse at the interface of the cell solution will generate cavitation bubbles. The shock wave can cause cell lysis. This technology is a non-contact, single-cell operation, high-efficiency, suitable for various cells, in vivo research, sterile environment, and can also be used in a laboratory-on-a-chip technology. The quantity requirements are relatively strict, and the required equipment is relatively expensive, the operation is complicated, and the cracking time is long, which is not conducive to wide application. ([9]Huang,S.-H.;Hung,L.-Y.;Lee,G.-B.Continuous nucleus extraction by optically-induced cell lysis on a batch-type microfluidic platform.Lab Chip 2016,16, 1447–1456. [10] Kremer, C.; Witte, C.; Neale, S.L.; Reboud, J.; Barrett, M.P.; Cooper, J.M. )

Electrolysis technology: The high-voltage electric field acts on the phospholipid bilayer of the cell membrane in the form of microsecond and millisecond pulses to generate an unstable potential, which will form a high-intensity transmembrane potential in the cell, resulting in a high-intensity potential, leading to cell lysis. This technology is a technology with high cell lysis efficiency and short time consumption, but requires high experimental conditions, expensive equipment and high heat production, which is not conducive to the extraction of temperature-sensitive intracellular components, thus greatly limiting its wide application. application. ([11] Ohshima, T.; Sato, M.; Saito, M. Selective release of intracellular protein using pulsed electric field. J. Electrost. 1995, 35, 103–112. [12] Ameri, S.K.; Singh, P.K.; Dokmeci , M.R.; Khademhosseini, A.; Xu, Q.; Sonkusale, S.R. All electronic approach for high-throughput cell trapping and lysis with electrical impedance monitoring. Biosens. Bioelectron. 2014, 54, 462–467.)

Acoustic lysis technology: a technology that realizes cell lysis with the help of sonic energy combined with the cavitation effect of microbubbles. Under the action of ultrasound, the microbubbles oscillate near the biological wall to generate microjets around them, and the shear force generated by the microjets on the biological wall can rupture the cell membrane. A relatively new method to separate and purify intracellular components by ultrasonic lysis of cells. The realization of sonication mainly depends on the cavitation effect of microbubbles. There are still some obstacles in the application and development of acoustic lysis technology due to the different sizes of cells that interact with microbubbles and the different acceptable shear stress thresholds of cells. Among them, due to the different ultrasonic radiation systems, the radiation dose calculation methods are diverse, and there is no relatively determined ultrasonic dose for cell lysis, and some parameters in the effect of ultrasonic radiation cannot be precisely controlled. At the same time, the cell lysis of the existing acoustic lysis technology still has the problems of high heat production and low cell lysis efficiency. ([12]Lentacker I, De Cock I, Deckers R. et al. Understanding ultrasound induced sonoporation: Definitions and underlying mechanisms [J]. Advanced Drug Delivery Reviews. 2014, 72: 49. [14] Study of a novel cell lysis method with titanium dioxide for lab-on-a-chip devices. Biomed. Microdevices 2011, 13, 527–532.)

SUMMARY OF THE INVENTION

In view of the above shortcomings, the present invention proposes a microfluidic chip cell lysis system based on a resonant microbubble array, which provides a miniature, single-cell, high-throughput, fast, and efficient cell lysis system, which generates heat Very low, simple operation, high reproducibility, can be used to extract and purify any components in cells, and can be widely used in various types of cells. This cell-free, cellular component-limited microfluidic system combines ultrasonic waves with microbubbles of uniform size. Based on the steady-state cavitation effect of microbubbles, it achieves fast, efficient, and highly stable cell lysis, and can be used in diseases. Demonstrate application prospects in diagnostics, gene therapy, cell engineering and drug screening.

One aspect of the present invention provides a device for lysing cells, the device comprising;

1) A cavity for forming a microbubble array, the cavity has a fluid channel, and two sides of the fluid channel are provided with microstructure cavities that communicate with the cavities and protrude outward, and the microstructure cavities can capture and hold microstructures. Bubble;

2) a substrate, which is bonded to the cavity, and jointly forms a fluid channel through which fluid can pass between the substrate and the fluid channel of the cavity;

3) a bulk wave generating device, which can generate bulk waves and transmit them into a cavity forming a microbubble array, forming resonance with the microbubbles;

In some embodiments of the invention, the bulk wave generating device is a bulk wave transducer. In a preferred embodiment, the frequency of the bulk wave transducer is 100-120 kHz, preferably 105-110 kHz, and in a specific embodiment, 107 kHz is used.

In some embodiments of the invention, the apparatus further includes a signal generator and a power amplifier.

In some embodiments of the present invention, the signal generator is configured to generate a sine wave signal and send the sine wave signal to the power amplifier.

In some embodiments of the present invention, the power amplifier is used for amplifying the sine wave signal, and sending the amplified sine wave signal to the bulk wave transducer.

In some embodiments of the present invention, the cavity forming the microbubble array has one or more fluid channels, and the microstructured cavities are staggered on both sides of the channel.

In some embodiments of the present invention, the size of the microstructure cavity is 1-100 μm in width and height, preferably 20-60 μm; preferably, the width and height of the microstructure cavity are 0.8 of the diameter of the cell to be lysed -1.2 times. In a preferred implementation of the present invention, the width of the microstructure cavity is 40.8 μm, and the height is 50 μm. In the technical solution of the present invention, the thickness of the microstructure cavity is higher than the cell diameter and less than 5 cm.

In some embodiments of the present invention, the cavity and the substrate are plasma bonded.

In some embodiments of the present invention, the material of the cavity is siloxane, preferably polydimethylsiloxane (PDMS).

In some embodiments of the invention, the cavity forming the microbubble array has at least one inlet and one outlet. In some preferred embodiments of the present invention, the inlet communicates with the liquid inlet device, and the outlet communicates with the cell lysate recovery device. In some preferred embodiments of the present invention, the liquid feeding device is a micro-injection pump. In some preferred embodiments of the present invention, the cell lysate recovery device is an analysis device, such as PCR equipment, cell analysis equipment, and the like.

Another aspect of the present invention provides a method for preparing the above-mentioned device, using photolithography to obtain a cavity for forming a microbubble array.

Another aspect of the present invention provides a method for lysing cells, viruses, bacteria or tissues, the method applies the above-mentioned device for lysing cells of the present invention, and includes the following steps:

1) placing a solution containing cells, viruses, bacteria or tissues in the cavity of the above-mentioned device, and the microstructure cavity of the cavity captures and produces microbubbles;

2) The body wave is generated by the body wave generating device in the microfluidic device, so that the microbubble resonates, causing the vibration of the solution in the cavity, and generating a flow field in the cavity for lysing cells or viruses .

In some embodiments of the invention, the solution comprising cells, viruses, bacteria or tissues in step 2) resides in the flow field generated by the body waves for at least 3 minutes.

In some embodiments of the present invention, step 3) recovery of lysed cells or virus liquid is also included.

In the technical solution of the present invention, the shear stress in the flow field around the microbubble is as follows

S=2π ^3/2 ε ² (ρ _f f ³ μ) ^1/2 /R _b ,

where R _b is the radius of the microbubble, _f is the resonant frequency of the microbubble, ρf is the density of the liquid, μ is the velocity of the relative fluid, and ε is the vibration amplitude of the microbubble.

Yet another aspect of the present invention provides the use of the above device of the present invention for cell lysis, bacterial lysis, virus lysis or tissue understanding.

beneficial effect

(1) The device of the present invention first has the characteristics of high throughput. The device of the present invention can make multiple channels, and in a specific embodiment, three rows of parallel channels are fabricated. Moreover, the side walls of each channel have equal and staggered microstructure cavities. The microstructure cavities of the present invention are arranged and staggered on both sides of the channel, and the flow fields generated by the vibration of different microbubbles interact independently. There will be a special flow field force between the flow fields, and this special flow field force can improve the lysis efficiency of cells, viruses, bacteria or tissues, and the vibration generated by the microbubbles in the microstructure cavity can act on the cells in the lumen.

(2) The microstructure cavity can capture microbubbles with the same radius. The array microbubbles vibrate at the same time under the excitation of the bulk wave generating device, and the amplitudes are the same. The second-order acoustic radiation force can trap the cells surrounding the microbubbles in the solution on the surface of the microbubbles. An equivalent shear stress is produced, causing cell lysis. Due to the equivalent shear stress, not only the lysis efficiency of cells is greatly improved, but also larger cell sample volumes can be processed at the same time.

(3) Accuracy, the cells are captured on the surface of the microbubble by the second-order acoustic radiation force, and the body wave can control the distance between the cells and the flow field, so as to accurately control the shear stress on the cells, so as to achieve the efficiency of cell lysis. precise control. The present invention only uses the body wave generating component, and does not use other components for controlling the position of the cells, thereby realizing the control of the position of the cells, capturing the cells on the surface of the microvesicles, and realizing efficient lysis.

(4) The device of the present invention has repeatability, the cavity can be processed by standard MEMS technology, and the device has good consistency, which further improves the repeatability of the experiment.

(5) The lysis method of the present invention has the characteristics of rapidity and simple operation, and it takes 30 minutes to realize the understanding of cells with a commercially available kit for lysing cells, but the device of the present invention can complete the lysis of cells in only 3 minutes, Moreover, when the outlet and inlet of the device are respectively coupled to the liquid inlet device and the cell lysis solution recovery device, continuous and uninterrupted cell lysis can be achieved, and increasing the number of cavities on the substrate can also increase the number of cells undergoing simultaneous lysis, compared to The lysis efficiency of commercial kits can be increased geometrically. In addition, the cleavage method of the present invention can achieve high efficiency without damaging DNA and proteins. For example, when the input voltage is 144Vpp, the lysis efficiency of MCF-7 cells can reach 97.6% after 1min of ultrasound. And it only takes 5 minutes to make a PDMS channel, and the PDMS structural template can be reused, and the entire experimental platform only takes 10 minutes to build.

(6) The present invention utilizes ultrasonic waves to regulate cell lysis, and the cell types are universal. It is only necessary to involve different cavities and microstructure cavities according to the size of the cells.

(7) The method of the present invention has the characteristic of producing extremely low heat, and can be used to extract and analyze temperature-sensitive proteins or enzymes.

Description of drawings

Figure 1 is a flow chart of the fabrication of the PDMS channel.

Figure 2 is a structural diagram of a PDMS channel.

Figure 3 is a schematic diagram of the structure of the experimental device.

Figure 4 is a flow field diagram generated by microbubble resonance at PDMS micropores.

Figure 5 shows the realization of the cell lysis effect observed under this system using propidium iodide (PI) and calcein-AM (Calcein-AM) double staining combined with fluorescence microscopy.

Detailed ways

In order to make the above objects, features and advantages of the present invention more clearly understood, the specific embodiments of the present invention will be described in detail below, but should not be construed as limiting the scope of the present invention.

Example 1 PDMS channel preparation and bonding

The fabrication process of PDMS is shown in Fig. 1(a-e).

(1) Pretreatment: The residual impurities on the surface of the silicon wafer, such as dust and organic adsorbates, are removed by pickling, alcohol washing and water washing, and finally the silicon wafer is placed in a clean place to dry.

(2) Glue coating and pre-baking: SU-8 (50) negative photoresist is spin-coated by a glue coating machine, 3000 rpm, 30 s, and the thickness of SU-8 (50) is about 50 μm. After applying the glue, place the silicon wafer horizontally on a 90°C heating plate for 1 hour to allow the solvent in the photoresist to volatilize to enhance the adhesion between the photoresist and the silicon wafer, and obtain a silicon wafer with a photoresist layer on the surface. , the structure is shown in Figure 1(a).

(3) Exposure and development: place a film with a pattern (as shown in (b) in Figure 1) on the side of the silicon wafer obtained in step (2) with a photoresist layer, and the film has a hollowed-out pattern, the hollow pattern is shown in Figure 2. Exposure was performed by an exposure machine pair with an exposure dose of 600 cJ/cm ² and a duration of 30 s. Soak the exposed silicon wafer with the developer solution, the photoresist layer in the unexposed area is dissolved, and the photoresist layer in the exposed area continues to remain. After developing, it is placed on a heating plate at 150 ° C and baked for 10 minutes to obtain the adhesion lithography on the silicon wafer. The photoresist layer is shaped like a hollowed-out pattern of a film, as shown in (d) of FIG. 1 .

(4) Casting PDMS: The A glue and B glue of PDMS are proportioned at a mass ratio of 10:1, mixed evenly, put into the petri dish where the silicon wafer obtained in step (3) is located, and the petri dish is evacuated to remove PDMS Finally, the culture dish was placed in an oven at 80 °C for 1 h to cure the PDMS. The structure of the obtained product is shown in Figure 1(e). The shape of the photoresist layer forms a negative groove in the PDMS layer.

(5) Peel off PDMS: use a scalpel to cut off the PDMS containing the pattern, and completely peel it off from the silicon wafer, and finally use a puncher to punch holes in the microchannels to make inlets and outlets.

Plasma treatment was performed on the PDMS channel and glass slide with a special structure. The power of the plasma treatment was 150W and the duration was 2 minutes. Then, the PDMS channel was pasted on the glass slide with the end down and baked in an oven at 80 °C for overnight.

Example 2 Construction of resonance microbubble array platform

The structure of the resonant microbubble array platform is shown in Figure 3. The experimental platform includes the following devices: signal generator, power amplifier, PDMS channel, microinjection pump, pipeline, cell recovery container, and body wave transducer.

in:

1. The signal generator provides a sine wave signal for the body wave transducer.

2. The power amplifier is to amplify the energy of the signal generated by the signal generator.

3. The microinjection pump can continuously inject liquid into the PDMS cavity.

4. The PDMS cavity contains an array of microstructures. When a liquid is injected into the PDMS cavity, there will be no inflow of liquid in the microstructure, thereby forming a micro-microbubble. The top view after injection is shown in Figure 5, which can be seen There is no liquid at the microstructure arrays on both sides of the channel, and microbubbles are formed.

5. Pipes are used to transfer liquids and solutions with cells.

6. The EP tube is used to return the liquid after cell lysis.

7. The body wave transducer is used to generate body waves. The body waves will cause the resonance of the microbubbles generated by the PDMS microstructure. The vibration of the microbubbles will cause the flow of the liquid in the liquid, and the shear force generated by the flow of the liquid will cause the cells to lyse.

Parameter selection and flow field characterization

As shown in Figure 4, due to the interface boundary effect, when liquid is injected into the PDMS channel, the micropores located on the sidewall of the PDMS channel will capture microbubbles. These arrayed microbubbles are excited by a specific frequency of a single ultrasonic source. The bubbles resonate and create a flow field. In the experiment, the width and height of the micro-holes on the sidewall of the PDMS channel are 40.8 μm and 50 μm in turn, the frequency of the bulk wave transducer is 107 kHz, and the input effective voltage value is 144 Vpp. Adding PS ball tracer particles into the PDMS cavity, it can be seen that under ultrasonic stimulation, the flow field around the microbubble is symmetrical and evenly distributed. And the shear stress in the flow field can be calculated by parameters such as the liquid velocity of the flow field. The formula for calculating shear stress is

S=2π ^3/2 ε ² (ρ _f f ³ )) ^1/2 /R _b ,

Example 3 Lysis cell experiment

Using the above resonance microbubble array platform of the present invention, the cell lysis experiment was carried out, and the cell lysis efficiency and the integrity of the DNA after cell lysis were quantitatively analyzed.

A solution containing cells labeled with two fluorescent probes, Calcein-AM and PI, was injected from the inlet using a microsyringe pump. The retention time of the cell-containing solution in the cavity was 3 minutes. And recover the lysate through the outlet, and analyze the lysis efficiency. Among them, Calcien-AM is a live cell indicator, which can freely penetrate the intact cell membrane and is hydrolyzed into calcein by intracellular esterase, which emits green fluorescence. When the cell membrane is damaged, PI can smoothly enter the cell and combine with DNA or RNA to emit red fluorescence. Combined with the cell sensitivity field map, the cell lysis efficiency can be calculated. It can be seen from the calculation that a lysis rate of 99% can be achieved when the cells remain in the channel for 3 minutes.

Claims

A device for cracking, characterized in that the device comprises;

1) A cavity for forming a microbubble array, the cavity has a fluid channel, and two sides of the fluid channel are provided with microstructure cavities that communicate with the cavities and protrude outward, and the microstructure cavities can capture and hold microstructures. Bubble;

2) a substrate, which is bonded to the cavity, and jointly forms a fluid channel through which fluid can pass between the substrate and the fluid channel of the cavity;

3) a bulk wave generating device, which can generate bulk waves and transmit them into a cavity forming a microbubble array, forming resonance with the microbubbles;

Preferably, the width and height of the microstructure cavity are respectively 1 μm-100 μm, preferably 40-50 μm.
The device according to claim 1, wherein the cavity for forming the microbubble array has at least one inlet and one outlet; preferably, the inlet is communicated with the liquid inlet device, and the outlet is connected with the cell lysate recovery device Unicom.
The device according to claim 1, wherein the bulk wave generating device is a bulk wave transducer; preferably, the frequency of the wave transducer is 100-120 kHz.
The apparatus of claim 1, wherein the apparatus further comprises a signal generator and a power amplifier.
The device according to claim 4, wherein the signal generator is configured to generate a sine wave signal and send the sine wave signal to the power amplifier.
The device according to claim 5, wherein the power amplifier is configured to amplify the sine wave signal, and send the amplified sine wave signal to the bulk wave transducer.
The device according to claim 1, wherein the cavity forming the microbubble array has one or more than two fluid channels, and the microstructure cavities are staggered on both sides of the channel.
A method for lysing cells, viruses, bacteria or tissues, wherein the method applies the device according to any one of claims 1-7, and comprises the following steps:

1) placing a fluid containing cells, viruses, bacteria or tissue in the lumen of the device, and the microstructure cavity of the lumen captures and produces microbubbles;

2) generating a body wave by a body wave generating device to resonate the microbubbles, causing the vibration of the solution in the cavity, and generating a flow field in the cavity for lysing cells or viruses;

Preferably, in the process of generating the body wave by the body wave generating device, the liquid containing cells, viruses, bacteria or tissues is continuously transported through the lumen.
The method according to claim 8, further comprising step 3) recovering the lysate.
Use of a device according to any one of claims 1 to 7 for cell lysis, bacterial lysis, viral lysis or tissue understanding.