Preparation method of microfluidic control device and viscoelastic-inertial microfluidic sorting device

Abstract

The present invention discloses a preparation method of a microfluidic control device and a viscoelastic-inertial microfluidic sorting device, which includes a stretching device and an ultra-stretched microfluidic control device detachably arranged on the stretching device; the stretching device includes a first square frame and a second square frame connected to each other, the first square frame is provided with an adjustable mounting mechanism for setting the ultra-stretched microfluidic control device, the second square frame is provided with an adjusting screw for adjusting the adjustable mounting mechanism, and one end of the adjusting screw is connected to one end of the adjustable mounting mechanism. The present invention can focus different biological particle species with a wide size distribution and various morphologies (such as spherical and spindle-shaped), and achieves separation of biological particles by adjusting the channel geometry, thereby realizing a pipeline that satisfies the sorting of biological particles with a wide range of diameters and different shapes.

Description

Technical Field

[0001] The present invention relates to the technical field of biological particle separation, and in particular to a preparation method of a microfluidic control device and a viscoelastic-inertial microfluidic sorting device. Background Art

[0002] How to select biological particles (bacteria, yeast, mammals, algae cells, etc.) is an important step in biological research. There is an urgent need to separate biological particles based on size and shape in a simpler, non-invasive and continuous manner. Microfluidics has the advantages of small sample volume, low cost, high precision and high efficiency. Viscoelastic-inertial microfluidics is considered to be an effective method to achieve three-dimensional particle separation. It focuses on simpler microfluidic channels without any external force fields or complex internal structures. In addition, this technology is suitable for relatively small particles (i.e., a few nanometers to submicron), which are usually difficult to handle in Newtonian fluid-based methods. Particle size is the basis of viscoelastic-inertial microfluidic sorting and separation. Since size-based separation is determined by the geometry of the microfluidic platform, once the conventional viscoelastic-inertial microfluidic device is prototyped, the channel size is difficult to change and only cells of a specific size can be sorted.

[0003] Traditional methods for separating bioparticles include streak plates, micropipettes, centrifugation, and membrane filtration. Streak plates are an old technique that can select morphologically distinct populations. However, algal cells produce similar green colonies, making them difficult to distinguish. While cells can be extracted using a micropipette under a light microscope, the process is laborious and low-throughput. Centrifugal elution and membrane filtration can be used to dehydrate algae, but they struggle to separate cells by size and require specialized and expensive equipment.

[0004] Several high-throughput, high-precision, and low-cost microfluidic separation methods have been investigated. The first is inertial microfluidic cell sorting. Active methods combine microfluidic flow with externally applied fields, such as electrical or magnetic fields, and therefore place higher demands on device fabrication and external components. Passive methods rely on microflows induced by specific channel designs or complex microstructural networks, such as deterministic lateral displacement (DLD) and hydrodynamic filtration (HDF). These particle manipulation methods are all performed in Newtonian fluids. Some techniques utilize external physical fields for bioparticle separation, such as electrophoresis, magnetophoresis, and acoustophoresis. However, external physical fields may provide additional stimulation to cells, and these methods generally require complex fabrication processes and bulky experimental setups. Inertial microfluidic cell sorting has low throughput, and the bioparticles that can be separated are large in diameter, which cannot meet the needs of sorting smaller particles (i.e., a few nanometers to submicrometers). Viscoelastic microfluidics is considered to be an effective way to achieve three-dimensional particles. It focuses in simpler microfluidic channels without any external force fields or complex internal structures. However, the focusing position of viscoelastic microfluidics in rectangular cross-section pipes is relatively complex and not conducive to observation. A simple way to reduce the focusing position is to moderately increase the flow rate so that the fluid inertia cannot be ignored and begins to affect the focusing behavior of the particles. This is called viscoelastic-inertial focusing.

[0005] Each viscoelastic-inertial microfluidic device has a certain separation threshold, which is mainly affected by the device size. To adjust the separation threshold and optimize separation performance, iterative design, fabrication, and testing are usually required, which is time-consuming, labor-intensive, and tedious.

[0006] Traditional enrichment and separation methods, such as centrifugal elution and membrane filtration, primarily separate cells by size. These methods require specialized and expensive equipment and large input volumes, making them unsuitable for the rare clinical sample situation. Integrating centrifugal elution into microfluidic devices has enabled the separation of cells within a range of diameters, but this approach suffers from low throughput and inefficient separation. Several low-cost, high-throughput, and high-precision microfluidic technologies have been developed for shape-based particle and cell separation. Some technologies, such as dielectrophoresis (DEP) and magnetophoresis, are based on integration with external application fields. However, these techniques often require complex fabrication processes and bulky external setups, and external actuation can adversely affect cells (e.g., cell damage from Joule heating). Other technologies rely on internal channels or microstructure-induced microfluidics, such as deterministic lateral displacement (DLD), hydrophoresis, and inertial focusing. However, DLD and hydrophoresis require complex microstructural networks (e.g., branched channels or arrays of pillars and grooves), resulting in high fabrication costs. Furthermore, once the device is prototyped, the channel size of typical viscoelastic-inertial microfluidic devices is difficult to change, limiting the separation of cells to a specific size. Summary of the Invention

[0007] The main purpose of the present invention is to propose a preparation method of a microfluidic control device and a viscoelastic-inertial microfluidic sorting device, aiming to achieve the ability to focus different biological particle species with a wide size distribution and various morphologies (such as spherical and spindle-shaped), and to achieve the separation of biological particles by adjusting the channel geometry.

[0008] To achieve the above objectives, the present invention provides a novel ultra-stretch viscoelastic-inertial microfluidic sorting device, comprising a stretching device and an ultra-stretch microfluidic control device detachably disposed on the stretching device.

[0009] The stretching equipment includes a first square frame and a second square frame connected to each other. The first square frame is provided with an adjustable mounting mechanism for setting the ultra-stretching microfluidic control device. The second square frame is provided with an adjusting screw for adjusting the adjustable mounting mechanism. One end of the adjusting screw is connected to one end of the adjustable mounting mechanism.

[0010] A further technical solution of the present invention is that the ultra-stretched microfluidic control device is in a bone shape.

[0011] A further technical solution of the present invention is that the ultra-stretched microfluidic control device includes a bone-shaped pipeline carrier and a microfluidic channel arranged on the central axis of the pipeline carrier.

[0012] A further technical solution of the present invention is that the bone-shaped pipeline carrier is made of Ecoflex ultra-stretchable flexible material, and the fluid pipeline is made of gallium wire as a sacrificial material.

[0013] A further technical solution of the present invention is that the adjustable mounting mechanism includes a first clamp and a second clamp arranged relatively to each other, the first clamp is fixedly arranged at one end of the first square frame, and the second clamp is movably arranged in the first square frame and connected to the adjusting screw.

[0014] A further technical solution of the present invention is that an adjusting knob is provided at the other end of the adjusting screw rod.

[0015] To achieve the above object, the present invention further provides a method for preparing an ultra-stretch microfluidic control device, which is applied to the novel ultra-stretch viscoelastic-inertial microfluidic sorting device as described above, and comprises the following steps:

[0016] Step S10, injecting liquid metal gallium into the hose, freezing the hose injected with gallium, and peeling off the outer skin of the frozen hose to obtain gallium filaments;

[0017] Step S20, pouring the uncured Ecoflex mixture onto the glass sheet with the bone-shaped acrylic plate attached thereto, and curing to form a thin layer of Ecoflex base;

[0018] Step S30: Fold the two ends of the prepared gallium filament by one micron and place it upright on the Ecoflex substrate, ensuring that the two ends of the upright filament are higher than the thickness of the acrylic board, and fill the acrylic board with the uncured Ecoflex mixture;

[0019] In step S40, after solidification, the gallium mold is melted at 40°C, and 0.1 mol / L sodium hydroxide solution is dripped onto both ends of the protruding gallium wire. As the sodium hydroxide solution continuously invades the gallium, the channel is gradually opened. Finally, a syringe filled with sodium hydroxide solution is aimed at one end of the channel and injected into the solution to flush out the remaining gallium in the pipeline. After flushing, a complete ultra-stretchable microfluidic control device is obtained.

[0020] The beneficial effects of the present invention's microfluidic control device preparation method and viscoelastic-inertial microfluidic sorting device are as follows: the present invention utilizes the phase change properties of metallic gallium, uses LM (gallium) wire as a sacrificial material, creates a three-dimensional microfluidic channel, and utilizes the ultra-stretching properties of Ecoflex to manufacture the channel carrier, achieving the ultra-stretching performance of this viscoelastic-inertial microfluidic device. This brings flexibility and stretchability to the viscoelastic-inertial microfluidic device, opening up new possibilities for large-scale modulation of channel geometry. This ultra-stretching viscoelastic-inertial microfluidic device can focus different bioparticle species with a wide size distribution and various morphologies (such as spherical and spindle shapes). By adjusting the channel geometry, the bioparticles can be separated, achieving a single channel to meet the needs of sorting bioparticles with a wide range of diameters and shapes. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. 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 structures shown in these drawings without paying any creative work.

[0022] Figure 1 2 is a schematic diagram of the overall structure of a preferred embodiment of the ultra-stretch viscoelastic-inertial microfluidic sorting device of the present invention;

[0023] Figure 2 It is a structural diagram of the stretching equipment;

[0024] Figure 3 is a schematic diagram of the structure of the ultra-stretchable microfluidic control device;

[0025] Figure 4 1 is a schematic flow chart of a method for preparing an ultra-stretched microfluidic control device according to the present invention;

[0026] Figure 5This is a comparative schematic diagram of the ultra-stretchable microfluidic control device before and after stretching;

[0027] Figure 6 This is an example of the successful sorting and enrichment of Haematococcus pluvialis using the ultra-stretch viscoelastic-inertial microfluidic sorting device.

[0028] The purpose, features and advantages of the present invention will be further described with reference to the accompanying drawings and in conjunction with the embodiments. DETAILED DESCRIPTION

[0029] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0030] Please refer to Figures 1 to 3 The present invention proposes a novel ultra-stretchable viscoelastic-inertial microfluidic sorting device. This device utilizes the phase change properties of metallic gallium and uses LM (gallium) wire as a sacrificial material to create a three-dimensional microfluidic channel 202. The ultra-stretchable properties of Ecoflex are then used to manufacture the channel carrier 201, achieving the ultra-stretchability of this viscoelastic-inertial microfluidic device. This brings flexibility and stretchability to the viscoelastic-inertial microfluidic device, opening up new possibilities for large-scale modulation of channel geometry. This ultra-stretchable viscoelastic-inertial microfluidic device is capable of focusing different bioparticle species with a wide size distribution and various morphologies (such as spherical and spindle shapes), and achieving bioparticle separation by adjusting the channel geometry.

[0031] Specifically, a preferred embodiment of the novel ultra-stretch viscoelastic-inertial microfluidic sorting device of the present invention includes a stretching device 10 and an ultra-stretch microfluidic control device 20 detachably disposed on the stretching device 10 .

[0032] The stretching device 10 includes a first square frame 101 and a second square frame 102 connected to each other. The first square frame 101 is provided with an adjustable mounting mechanism for setting the ultra-stretching microfluidic control device 20. The second square frame 102 is penetrated by an adjusting screw 105 for adjusting the adjustable mounting mechanism. One end of the adjusting screw 105 is connected to one end of the adjustable mounting mechanism.

[0033] Specifically, in this embodiment, the ultra-stretched microfluidic control device 20 is in a bone shape.

[0034] More specifically, in this embodiment, the ultra-stretched microfluidic control device 20 includes a bone-shaped channel carrier 201 and a microfluidic channel 202 arranged on the central axis of the channel carrier.

[0035] When the length of the mounting mechanism is extended by rotating the adjusting screw 105 , the geometric shape of the fluid channel in the bone-shaped pipe changes simultaneously, thereby achieving separation of biological particles by adjusting the geometric shape of the microfluidic channel 202 .

[0036] The adjustable mounting mechanism includes a first clamp 103 and a second clamp 104 arranged opposite to each other. The first clamp 103 is fixedly arranged at one end of the first square frame 101, and the second clamp 104 is movably arranged in the first square frame 101 and connected to the adjusting screw rod 105.

[0037] In this embodiment, one end of the ultra-stretch microfluidic control device 20 is connected to the first clamp 103, and the other end is connected to the second clamp 104. When the adjusting screw 105 is rotated to make the second clamp 104 move away from the first clamp 103, the distance between the first clamp 103 and the second clamp 104 increases, thereby causing the geometric shape of the fluid channel to change. In this way, the separation of biological particles is achieved by adjusting the geometric shape of the microfluidic channel 202.

[0038] Furthermore, in this embodiment, the bone-shaped pipeline carrier 201 is made of Ecoflex ultra-stretchable flexible material, and the fluid pipeline is made of gallium wire as a sacrificial material.

[0039] The present invention uses LM (gallium) wire as a sacrificial material to create a three-dimensional microfluidic channel 202, and uses the ultra-stretchable material Ecoflex to make a microfluidic device. The channel diameter is changed by stretching (the tensile strain can reach more than 500%) to adapt to the focusing of cells of different diameters and shapes. At the same time, welding is used to change the shape of the gallium wire to manufacture different microfluidic devices to achieve the purpose of sorting and enriching cells.

[0040] It is worth mentioning that, in this embodiment, the Ecoflex ultra-stretchable flexible material may also be replaced by materials with similar functions such as hydrogel, and the present invention is not limited to this.

[0041] Furthermore, in this embodiment, an adjusting knob 106 is provided at the other end of the adjusting screw rod 105. In this embodiment, the adjusting knob 106 is provided at the other end of the adjusting screw rod 105, which facilitates user operation.

[0042] The beneficial effects of the novel ultra-stretch viscoelastic-inertial microfluidic sorting device of the present invention are as follows: the present invention, through the above-mentioned technical scheme, includes a stretching device and an ultra-stretch microfluidic control device detachably arranged on the stretching device; the stretching device includes a first square frame and a second square frame connected to each other, an adjustable mounting mechanism for setting the ultra-stretch microfluidic control device is provided in the first square frame, and an adjusting screw for adjusting the adjustable mounting mechanism is passed through the second square frame, one end of the adjusting screw is connected to one end of the adjustable mounting mechanism, and the phase change characteristics of metallic gallium are utilized, LM (gallium) wire is used as a sacrificial material to create a three-dimensional microfluidic channel, and the Ecoflex ultra-stretching characteristics are utilized to manufacture a pipeline carrier, thereby realizing the ultra-stretching performance of this viscoelastic-inertial microfluidic device, bringing flexibility and stretchability to the viscoelastic-inertial microfluidic device, and opening up new possibilities for large-scale modulation of channel geometry. This ultra-stretch viscoelastic-inertial microfluidic device can focus different biological particle species with a wide size distribution and various morphologies (such as spherical and spindle-shaped). By adjusting the channel geometry, the separation of biological particles is achieved, and a single channel can be used to separate biological particles with a wide range of diameters and different shapes. In addition, the present invention has a simple structure and is easy to operate.

[0043] To achieve the above object, the present invention also proposes a method for preparing an ultra-stretched microfluidic control device, which is applied to the novel ultra-stretched viscoelastic-inertial microfluidic sorting device as described above. Figure 4 As shown, the method includes the following steps:

[0044] Step S10: injecting liquid metal gallium into the hose, freezing the hose injected with gallium, and peeling off the outer skin of the frozen hose to obtain gallium filaments.

[0045] Specifically, in this embodiment, liquid metal gallium is first injected into the hose, the hose injected with gallium is frozen, and the outer skin of the frozen hose is peeled off with a scalpel to obtain a gallium filament with a diameter of 100 μm or 300 μm (the diameter depends on the hose diameter).

[0046] In step S20, uncured Ecoflex mixture (Ecoflex 00-31) is poured onto the glass sheet with the bone-shaped acrylic plate attached thereto, and cured to form a thin layer of Ecoflex base.

[0047] In step S30 , the two ends of the prepared gallium wire are folded up by one micron and placed vertically on the Ecoflex substrate, ensuring that the two ends of the vertical wire are higher than the thickness of the acrylic board, and the acrylic board is fully filled with the uncured Ecoflex mixture.

[0048] In step S40, after solidification, the gallium mold is melted at 40°C. A 0.1 mol / L sodium hydroxide solution is dripped onto both ends of the protruding gallium filaments. As the sodium hydroxide solution continuously penetrates the gallium, the channel is gradually opened. Finally, a syringe filled with sodium hydroxide solution is injected into one end of the channel to flush out the remaining gallium. After flushing, the ultra-stretchable microfluidic control device is completed. The total length of the channel is 28 mm.

[0049] like Figure 5 As shown, Figure 5 A comparative schematic diagram of the ultra-stretch microfluidic control device before and after stretching is depicted. The microfluidic device (chip) is mounted on a customized stretching device. After fixing both ends of the chip on the stretching device, rotating the knob can cause the device to drive the chip to stretch, thereby changing the chip pipeline length. To complete the sorting of biological particles, a syringe pump is first used to introduce the particles or cell suspension into the ultra-stretch viscoelastic-inertial microfluidic device to maintain a fixed flow rate between 1-600μL / min. The entire device is then placed on the platform of an inverted microscope to monitor and record the flow of the sample in the microchannel. The microfluidic chip is stretched over a large range through the stretching device (a maximum stretching amount of 500% is achievable), and the diameter of the microfluidic pipeline can be changed to adjust the focus of cells over a large range.

[0050] The following combination Figure 6 The experimental process and effects of the ultra-stretched microfluidic control device of the present invention are described.

[0051] Figure 6 This is an example of the successful sorting and enrichment of Haematococcus pluvialis using the ultra-stretch viscoelastic-inertial microfluidic device.

[0052] Because the migration of Haematococcus pluvialis cells can be regulated by stretching the Ecoflex channel, we used Ecoflex extension to achieve sheath-free, label-free, and continuous separation or enrichment of Haematococcus pluvialis cells. With continued stretching of the Ecoflex channel, at an Ecoflex strain of 100%, large cells tend to migrate toward the channel centerline and exit through the middle outlet 1 (O1'), while small cells tend to exit through the side outlet 2 (O2'). We call this the sorting mode; with further extension (~200%), both small and large cells tend to migrate toward the channel centerline and exit through the middle outlet 1 (O1"), representing the enrichment mode, which yields a higher concentration of Haematococcus pluvialis cells.

[0053] The beneficial effects of the method for preparing the ultra-stretchable microfluidic control device of the present invention are as follows: the ultra-stretchable microfluidic control device prepared by the present invention through the above-mentioned technical solution utilizes the phase change properties of metallic gallium and uses LM (gallium) wire as a sacrificial material to create a three-dimensional microfluidic channel. The ultra-stretchable properties of Ecoflex are utilized to manufacture the channel carrier to achieve the ultra-stretchable performance of this viscoelastic-inertial microfluidic device, which brings flexibility and stretchability to the viscoelastic-inertial microfluidic device and opens up new possibilities for large-scale modulation of channel geometry. This ultra-stretchable viscoelastic-inertial microfluidic device can focus different bioparticle species with a wide size distribution and various morphologies (such as spherical and spindle-shaped). By adjusting the channel geometry, the separation of bioparticles is achieved, and a single channel can meet the needs of sorting bioparticles with a wide range of diameters and different shapes.

[0054] The above description is only a preferred embodiment of the present invention and does not limit the patent scope of the present invention. All equivalent structural transformations made by using the contents of the present invention description and drawings under the concept of the present invention, or direct / indirect application in other related technical fields are included in the patent protection scope of the present invention.

Claims

1. An ultra-stretch viscoelastic-inertial microfluidic sorting device, characterized in that: It comprises a stretching device and an ultra-stretching microfluidic control device detachably arranged on the stretching device; The stretching device includes a first square frame and a second square frame connected to each other, wherein an adjustable mounting mechanism for mounting the ultra-stretched microfluidic control device is provided in the first square frame, and an adjusting screw for adjusting the adjustable mounting mechanism is passed through the second square frame, and one end of the adjusting screw is connected to one end of the adjustable mounting mechanism; The ultra-stretched microfluidic control device includes a bone-shaped pipeline carrier and a microfluidic channel arranged on the central axis of the pipeline carrier; The bone-shaped channel carrier is made of Ecoflex ultra-stretchable flexible material, and the microfluidic channel is made of gallium wire as a sacrificial material; The adjustable mounting mechanism includes a first clamp and a second clamp that are arranged opposite to each other. The first clamp is fixedly arranged at one end of the first square frame, and the second clamp is movably arranged in the first square frame and connected to the adjusting screw rod.

2. The ultra-stretch viscoelastic-inertial microfluidic sorting device according to claim 1, characterized in that: The other end of the adjusting screw rod is provided with an adjusting knob.

3. A method for preparing an ultra-stretched microfluidic control device, characterized in that: The preparation method is applied to the ultra-stretch viscoelastic-inertial microfluidic sorting device according to any one of claims 1 to 2, and the method comprises the following steps: Step S10, injecting liquid metal gallium into the hose, freezing the hose injected with gallium, and peeling off the outer skin of the frozen hose to obtain gallium filaments; Step S20, pouring the uncured Ecoflex mixture onto the glass sheet with the bone-shaped acrylic plate attached thereto, and curing to form a layer of Ecoflex base; Step S30: Fold the two ends of the prepared gallium filament by one micron and place it upright on the Ecoflex substrate, ensuring that the two ends of the upright filament are higher than the thickness of the acrylic board, and fill the acrylic board with the uncured Ecoflex mixture; In step S40, after solidification, the gallium mold is melted at 40°C, and 0.1 mol / L sodium hydroxide solution is dripped onto both ends of the protruding gallium wire. As the sodium hydroxide solution continuously invades the gallium, the channel is gradually opened. Finally, a syringe filled with sodium hydroxide solution is aimed at one end of the channel and injected into the solution to flush out the remaining gallium in the pipeline. After flushing, a complete ultra-stretchable microfluidic control device is obtained.

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