CN117483018A

CN117483018A - Biological particle separating device, processing method and microfluidic chip

Info

Publication number: CN117483018A
Application number: CN202311482223.1A
Authority: CN
Inventors: 王文会; 柴惠超
Original assignee: Tsinghua University
Current assignee: Tsinghua University
Priority date: 2023-10-17
Filing date: 2023-11-08
Publication date: 2024-02-02

Abstract

The invention discloses a biological particle separating device, a processing method and a micro-fluidic chip, wherein the biological particle separating device comprises: a microchannel comprising a particle suspension inlet, a focusing sheath liquid inlet, a sample channel, a particle suspension outlet, a focusing sheath liquid outlet, the particle suspension inlet and the focusing sheath liquid inlet communicating the particle suspension outlet and the focusing sheath liquid outlet via the sample channel; the liquid metal inlet is communicated with the liquid metal outlet through the electrode runner, and the electrode runner is used for forming at least one group of three-dimensional microelectrodes with opposite polarities; and the substrate is used for fixing the micro-flow channel, the liquid metal inlet, the electrode flow channel and the liquid metal outlet. The biological particle separation device disclosed by the embodiment of the disclosure realizes continuous, progressive and high-flux dielectrophoresis biological particle separation.

Description

Biological particle separating device, processing method and microfluidic chip

Technical Field

The invention relates to the technical field of microfluidics, in particular to a biological particle separation device, a processing method and a microfluidic chip.

Background

The inclusion of particulates, cells, droplets and other contaminating particles in complex fluid samples has become a critical issue in many fields of application, such as industrial applications, environmental assessment, biochemical analysis, and the like.

In the related art, biomarker-based methods are the main tool for the separation of biological particles, which recognize specific populations of biological particles by labeling biological particles (e.g., cells) with molecular-specific labels (e.g., fluorescent dyes, quantum dots, magnetic beads, stable isotopes, etc.). Labeling of biological particles not only requires a priori knowledge of the specificity of the biological particles, but is also limited by the expensive equipment, the time consuming and potential impact of the labeling on downstream analysis.

In addition, the related art performs the separation of the biological particles by using a label-free method, in contrast, the label-free method realizes the separation of the biological particles by identifying and utilizing the inherent characteristics of the biological particles, such as size, density, biophysical characteristics, and the like, avoids the use of external labels, thereby being easier to maintain the integrity and bioactivity of the biological particles, and can realize the noninvasive, high-throughput and efficient separation of the biological particles. This makes it a potential tool for biological particle separation and research, which is of great interest. The label-free biological particle separation method includes, for example, density gradient centrifugation, gravity sedimentation, filtration membrane separation, and the like. These methods are often used to separate target biological particles from heterogeneous biological fluid samples, and are mostly excellent in terms of processing throughput, etc., helping researchers to understand and recognize biological particles and greatly improving, however, these methods have problems of low purity, recovery rate and biological activity, which limits their applicability for further analysis and evaluation.

In contrast to the above approach, dielectrophoresis microfluidic techniques can separate biological particles based on their size and dielectric properties, providing accurate, fast, low cost and label-free biological particle manipulation capability. However, due to the limited scope of dielectrophoresis force, the biological particle separation method based on dielectrophoresis microfluidic technology cannot provide enough dielectrophoresis force action time when processing samples with high flow rate, and the processing flux is low.

Disclosure of Invention

In view of this, the present disclosure proposes a technical solution for biological particle separation.

In one possible implementation, the apparatus includes: a microchannel comprising a particle suspension inlet, a focusing sheath liquid inlet, a sample channel, a particle suspension outlet, a focusing sheath liquid outlet, the particle suspension inlet and the focusing sheath liquid inlet communicating the particle suspension outlet and the focusing sheath liquid outlet via the sample channel; the liquid metal inlet is communicated with the liquid metal outlet through the electrode runner, and the electrode runner is used for forming at least one group of three-dimensional microelectrodes with opposite polarities; and the substrate is used for fixing the micro-flow channel, the liquid metal inlet, the electrode flow channel and the liquid metal outlet.

In one possible implementation, the electrode runner includes capillary valves having different trigger pressure thresholds for controlling the flow direction of the liquid metal in the electrode runner.

In one possible implementation, the capillary valve includes a stop valve, a passive switching valve, the electrode flow channel is communicated with the sample flow channel through the stop valve, a first path of the electrode flow channel is communicated with a branched second path through the passive switching valve, a cross-sectional area of the electrode flow channel is larger than a cross-sectional area of the passive switching valve, and a cross-sectional area of the passive switching valve is larger than a cross-sectional area of the stop valve.

In one possible implementation, the shut-off valve is configured to prevent the liquid metal from entering the sample flow path after filling the first path of the electrode flow path; the passive switching valve is used for: and in case the first path of the electrode runner is not filled with liquid metal, preventing the liquid metal from entering the branched second path, and in case the first path of the electrode runner is filled with liquid metal, switching the flow direction of the liquid metal from the first path to the branched second path.

In one possible implementation manner, the electrode flow channel is provided with an asymmetric electrode, and each stop valve provided on the positive electrode side corresponds to a plurality of stop valves provided on the negative electrode side, or each stop valve provided on the negative electrode side corresponds to a plurality of stop valves provided on the positive electrode side.

In one possible implementation, the particle suspension inlet and the particle suspension outlet are connected to a microfluidic pump by means of a plastic hose, in order to control the flow of the particle suspension in the microfluidic channel by means of the microfluidic pump.

In one possible implementation, the liquid metal includes at least one of indium, tin, cadmium, bismuth, lead, gallium.

According to another aspect of the present disclosure, there is provided a method of processing a biological particle separation device, the method comprising: manufacturing the biological particle separating device through a soft lithography process; and injecting liquid metal into a liquid metal inlet of the biological particle separating device, and integrating the electrode pattern self-assembled by the biological particle separating device into a large-array three-dimensional microelectrode by utilizing capillary valves with different trigger pressure thresholds.

In one possible implementation, the capillary valve includes a stop valve, a passive switching valve, the electrode flow channel is communicated with the sample flow channel through the stop valve, a first path of the electrode flow channel is communicated with a branched second path through the passive switching valve, a cross-sectional area of the electrode flow channel is larger than a cross-sectional area of the passive switching valve, and a cross-sectional area of the passive switching valve is larger than a cross-sectional area of the stop valve; the self-assembly process of the biological particle separation device comprises the following steps: when the liquid metal is injected into the electrode runner and flows to the path branches, the passive switching valve blocks the liquid metal from entering the second paths of the branches, and the liquid metal flows forwards along the first paths; the stop valve prevents liquid metal from entering the sample runner under the condition that the liquid metal fills the first path of the electrode runner, and the passive switching valve switches the flow direction of the liquid metal from the first path to the branched second path; and when the liquid metal enters the branched second path, taking the branched second path as the first path of the trunk, and repeatedly executing the process until the flowing liquid metal fills the electrode runner to form an electrode pattern.

According to another aspect of the present disclosure, there is provided a microfluidic chip including the biological particle separation device as described above.

In an embodiment of the present disclosure, a biological particle separation device includes: a microchannel comprising a particle suspension inlet, a focusing sheath liquid inlet, a sample channel, a particle suspension outlet, a focusing sheath liquid outlet, the particle suspension inlet and the focusing sheath liquid inlet communicating the particle suspension outlet and the focusing sheath liquid outlet via the sample channel; the liquid metal inlet is communicated with the liquid metal outlet through the electrode runner, and the electrode runner is used for forming at least one group of three-dimensional microelectrodes with opposite polarities; and the substrate is used for fixing the micro-flow channel, the liquid metal inlet, the electrode flow channel and the liquid metal outlet. In this way, a large array of three-dimensional microelectrodes based on liquid metal can be integrated in a compact micro-channel, and a larger range of action and longer action time dielectrophoresis force are provided for controlling biological particles by generating a large number of electric field gradients in series in the micro-channel, so that continuous, progressive and high-flux separation of biological particles is realized.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the disclosure. Other features and aspects of the present disclosure will become apparent from the following detailed description of exemplary embodiments, which proceeds with reference to the accompanying drawings.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the disclosure and together with the description, serve to explain the technical aspects of the disclosure.

Fig. 1 shows a schematic structural view of a biological particle separating apparatus according to an embodiment of the present disclosure.

Fig. 2 shows a schematic diagram of a bio-particle separation device integrating a large array of three-dimensional microelectrodes according to embodiments of the present disclosure.

Fig. 3 shows a schematic diagram of the capillary valve principle of an embodiment of the present disclosure.

Fig. 4 shows a schematic structural view of an electrode flow channel according to an embodiment of the present disclosure.

Fig. 5 shows a schematic diagram of a liquid metal electrode array self-assembly workflow of an embodiment of the present disclosure.

Fig. 6 shows a schematic diagram of a uniform sphere model and a single shell sphere model of an embodiment of the present disclosure.

Fig. 7 shows a schematic diagram of electric field strength simulation of a three-dimensional microelectrode according to an embodiment of the present disclosure.

Fig. 8 shows a schematic diagram of a validation experiment of a biological particle separation device of an embodiment of the disclosure.

Fig. 9 shows a schematic diagram of another validation experiment of a biological particle separation device of an embodiment of the disclosure.

Detailed Description

Various exemplary embodiments, features and aspects of the disclosure will be described in detail below with reference to the drawings. In the drawings, like reference numbers indicate identical or functionally similar elements. Although various aspects of the embodiments are illustrated in the accompanying drawings, the drawings are not necessarily drawn to scale unless specifically indicated.

The word "exemplary" is used herein to mean "serving as an example, embodiment, or illustration. Any embodiment described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments.

The term "and/or" is herein merely an association relationship describing an associated object, meaning that there may be three relationships, e.g., a and/or B, may represent: a exists alone, A and B exist together, and B exists alone. In addition, the term "at least one" herein means any one of a plurality or any combination of at least two of a plurality, for example, including at least one of A, B, C, and may mean including any one or more elements selected from the group consisting of A, B and C.

Furthermore, numerous specific details are set forth in the following detailed description in order to provide a better understanding of the present disclosure. It will be understood by those skilled in the art that the present disclosure may be practiced without some of these specific details. In some instances, methods, means, elements, and circuits well known to those skilled in the art have not been described in detail in order not to obscure the present disclosure.

Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to like or similar elements or elements having like or similar functions throughout. The embodiments described below by referring to the drawings are illustrative and intended to explain the present invention and should not be construed as limiting the invention.

Microfluidic technology (Microfluidics) can process or manipulate micro-fluids (with a size of tens to hundreds of micrometers) using micro-channels (with a volume of nano liter to attic liter), and has advantages in terms of system integration, such as small sample volume, strong integration capability, good biocompatibility, and fast response speed.

In order to improve the sample processing throughput of dielectrophoresis microfluidic devices, in the related art, planar electrodes may be replaced with three-dimensional electrodes in the devices. For example, a microfluidic device using a conductive ionic liquid as a three-dimensional electrode on a side wall can use the surface tension of the liquid to maintain the position of the conductive ionic liquid, and apply dielectrophoresis force to biological particles passing through a sample channel. For another example, a microfluidic device using a conductive polymer formed by mixing silver-polydimethylsiloxane as a three-dimensional staggered electrode can induce biological particle separation using dielectrophoresis forces generated by staggered electrode tracks. These methods are easy to manufacture three-dimensional electrodes in microfluidic devices, but the electrode materials used are low in conductivity, affecting the electric field coupling, limiting their applicability to high-throughput separation scenarios.

The liquid metal alloy combines excellent electrical conductivity with good fluidity, making it a target material for constructing three-dimensional electrodes in high-throughput microfluidic devices. The injection of the liquid metal alloy into the micro-fluidic channel can produce a simple three-dimensional contact electrode aligned with the channel and generate a strong and uniform three-dimensional electric field in the micro-fluidic channel. For example, in the related art, the electrode flow path may be separated from the sample flow path by providing a microcolumn barrier in the microcolumn flow path, preventing the liquid metal from flowing into the sample flow path. And then liquid metal is injected into the designed electrode flow channel as liquid at a certain temperature, and when the liquid metal alloy is recovered to room temperature and solidified, a self-assembled three-dimensional electrode array is formed between microcolumns at two sides of the sample flow channel. Such methods of co-fabrication of liquid metal electrodes and sample channels lack the ability to accurately control the flow of liquid metal, typically involve only the simplest patterns of straight lines and curves, and have low space utilization, which inevitably results in an impractical expansion of chip size in the face of high throughput scenarios where as many electrodes as possible are required.

Therefore, in the task of sample separation, the microfluidic device using the ionic liquid and the conductive polymer as electrode materials has low conductivity due to the electrode materials, influences the coupling efficiency of the electric field flow field, cannot apply enough dielectrophoresis force to the sample at high flow speed, and limits the improvement of flux. The device using the liquid metal alloy as the electrode material lacks a method for accurately controlling the flow of the liquid metal, and cannot construct electrodes with complex patterns, so that it is difficult to integrate large-array three-dimensional liquid metal electrodes in a microfluidic chip with limited size, and a limited number of electrodes cannot provide enough dielectrophoresis force action time for a high-flow-rate sample, so that the sample processing flux is difficult to promote.

In order to improve the capability of unmarked separation of particles in a high-flux scene, the embodiment of the disclosure provides a biological particle separation device, which can integrate a large array of three-dimensional microelectrodes based on liquid metal in a compact micro-channel, and provide a larger action range and a longer action time dielectrophoresis force for biological particle manipulation by generating a large number of electric field gradients in series in the micro-channel, so as to realize continuous, progressive and high-flux biological particle separation.

Fig. 1 shows a schematic structural diagram of a biological particle separation device according to an embodiment of the present disclosure, as shown in fig. 1, the biological particle separation device includes a micro flow channel (see blue part of fig. 1) including a particle suspension inlet 101, a focusing sheath liquid inlet 102, a sample flow channel 103, a particle suspension outlet 104, and a focusing sheath liquid outlet 105, the particle suspension inlet 101 and the focusing sheath liquid inlet 102 communicating the particle suspension outlet 104 and the focusing sheath liquid outlet 105 via the sample flow channel 103.

The biological particle separating device comprises a liquid metal inlet, an electrode runner and a liquid metal outlet (see yellow part of fig. 1), wherein the liquid metal inlet is communicated with the liquid metal outlet through the electrode runner. As shown in fig. 1, the liquid metal inlet 201 communicates with the liquid metal outlet 202 via an electrode runner 206; the liquid metal inlet 203 communicates with the liquid metal outlet 204 via an electrode runner 205. The polarities of the liquid metal inlet 201, the electrode runner 206 and the liquid metal outlet 202 are the same, the polarities of the liquid metal inlet 203, the electrode runner 205 and the liquid metal outlet 204 are the same, and the polarities of the liquid metal inlet 201, the electrode runner 206 and the liquid metal outlet 202 are opposite (or opposite) to the polarities of the liquid metal inlet 203, the electrode runner 205 and the liquid metal outlet 204. The electrode runners 205 and the electrode runners 206 can be used to form at least one set of three-dimensional microelectrodes of opposite polarity, the number of which is positively correlated to the length of the electrode runners.

The biological particle separation device further comprises a substrate 301 for fixing the micro flow channel, the liquid metal inlet, the electrode flow channel and the liquid metal outlet.

In one possible implementation, the polarity of the electrode runners may be set by applying electrical signals of different polarities to the electrode runners due to the conductivity of the metal. In order to make the polarities of the two electrode runners 205 and the electrode runner 206 on both sides of the sample runner 103 different, the polarities of the liquid metal inlet 201, the electrode runner 206, and the liquid metal outlet 202 may be set as positive electrodes, and the polarities of the liquid metal inlet 203, the electrode runner 205, and the liquid metal outlet 204 may be set as negative electrodes; the polarities of the liquid metal inlet 201, the electrode runner 206, and the liquid metal outlet 202 may be set as a negative electrode, and the polarities of the liquid metal inlet 203, the electrode runner 205, and the liquid metal outlet 204 may be set as a positive electrode, which is not limited in the present disclosure.

In one possible implementation, the electrode channels with opposite polarities are embedded in the micro-channel, and in the case that two opposite polarities of the electrode channel 205 and the electrode channel 206 are disposed on two sides of the sample channel 103 of the micro-channel, the shapes of the sample channel 103, the electrode channel 205 and the electrode channel 206 can be set to be various shapes such as a circle, a trapezoid, a rectangle or a square, and the shapes and the sizes of the sample channel 103, the electrode channel 205 and the electrode channel 206 can be matched according to the particle size to be tested, which is not limited in the disclosure.

In the example shown in fig. 1, the sample flow channel 103 is arranged in a zigzag shape in a portion between the particle suspension inlet 101, the focusing sheath liquid inlet 102, and the particle suspension outlet 104, and the focusing sheath liquid outlet 105, the number of turning back and forth is not limited, and accordingly, the electrode flow channel 205 and the electrode flow channel 206 may be in a comb shape, and each have an extension portion (for example, a first extension portion 205-1, a second extension portion 205-2, and a third extension portion 205-3 corresponding to the electrode flow channel 205; a first extension portion 206-1, a second extension portion 206-2, and a third extension portion 206-3 corresponding to the electrode flow channel 206) in a comb shape, and the gap extending to the sample flow channel 103 forms a structure similar to an "finger-inserting", where the electrode flow channel 205 and the electrode flow channel 206 having opposite polarities are always present on both sides of the sample flow channel 103, that is, the electrode flow channel 205 is on one side, and the electrode flow channel 206 is on the other side. In the example shown in fig. 1, the liquid metal inlet 203, the liquid metal outlet 204 are located on a first side of the substrate 301, the liquid metal inlet 201, the liquid metal outlet 202 are located on a second side opposite to the first side, the particle suspension outlet 104, the focusing sheath liquid outlet 105 are located on a third side adjacent to the first side and near the liquid metal inlet 203, the liquid metal outlet 204, the particle suspension inlet 101, the focusing sheath liquid inlet 102 are located on a fourth side opposite to the third side and near the liquid metal inlet 201, the liquid metal outlet 202. Wherein, it should be understood by those skilled in the art that the specific positions and shapes of the above components can be changed as required, and the present application is not limited thereto.

Similarly, embodiments of the present disclosure provide for particle suspension inlet 101, focusing sheath fluid inlet 102, particle suspension outlet 104, focusing sheath fluid outlet 105, oppositely polarized liquid metal inlet 201 and liquid metal inlet 203, oppositely polarized liquid metal outlet 202 and liquid metal outlet 204, the shape and size of which are not particularly limited and may be flexibly configured according to the actual use scenario.

The number of the three-dimensional microelectrodes formed is related to the length of the electrode runner, and the longer the length of the electrode runner, the more number of the three-dimensional microelectrodes can be formed, in practical application, the number of the three-dimensional microelectrodes can be infinitely increased, and according to the number of the three-dimensional microelectrodes, the length of the sample runner 103 can be correspondingly increased.

In one possible implementation, the liquid metal includes at least one of indium, tin, cadmium, bismuth, lead, gallium. The electrode flow channel can be filled with a liquid metal simple substance or a liquid metal alloy, for example, a liquid metal composed of indium, tin, cadmium, bismuth and lead alloy, a liquid metal composed of gallium simple substance and a liquid metal composed of gallium indium alloy, and the specific constituent materials of the liquid metal are not limited in the disclosure.

In one possible implementation, embodiments of the present disclosure also provide a microfluidic chip including a biological particle separation device as shown in fig. 1.

Fig. 2 is a schematic diagram illustrating integration of a large array of three-dimensional microelectrodes in a biological particle separation device according to an embodiment of the present disclosure, as shown in fig. 2, a microfluidic chip shown in section b is used as an example of the biological particle separation device, and the microfluidic chip adopts the structure shown in fig. 1, and after power is applied, a large number of (for example 5000 pairs of) three-dimensional liquid metal electrodes are integrated in the chip, and an equivalent structure of each pair of three-dimensional microelectrodes is shown as a section 401 in the drawing, and equivalent connection between each pair of three-dimensional microelectrodes is shown as a section c.

It should be understood that the spacing between any two adjacent pairs of three-dimensional microelectrodes 401 may be set to any value, for example, as shown in fig. 2, along the sample flow path 103, the spacing L1 between the first pair of three-dimensional microelectrodes 401 and the second pair of three-dimensional microelectrodes 401, the spacing L2 between the second pair of three-dimensional microelectrodes 401 and the third pair of three-dimensional microelectrodes 401, and the spacing L3 between the second pair of three-dimensional microelectrodes 401 and the third pair of three-dimensional microelectrodes 401 may be set to the same value, or L1, L2, L3 may be set to different values, and embodiments of the present disclosure do not limit the spacing between the three-dimensional microelectrodes 401.

In one possible implementation, the sample channel 103 and the electrode channels (e.g., electrode channel 205 and electrode channel 206 with opposite polarities) are fabricated by a soft lithography process and formed by a Polydimethylsiloxane (PDMS) reverse mold.

Alternatively, the liquid metal may be self-assembled after patterning by pushing or other mechanical means (including, for example, using a microfluidic pump, linear motor, etc.) to drive a syringe into the electrode runner.

Optionally, the particle suspension inlet 101 and the particle suspension outlet 104 are connected to a microfluidic pump through a plastic hose, so as to control the flow of the particle suspension in the microfluidic channel through the microfluidic pump, and sample injection can also be implemented by sucking a syringe at the particle suspension outlet 104.

Optionally, the focusing sheath fluid inlet 102 and the focusing sheath fluid outlet 105 may be connected to a microfluidic pump through a plastic hose, so as to control the flow of the focusing sheath fluid in the microfluidic channel through the microfluidic pump, and sample injection may be implemented by sucking a syringe at the focusing sheath fluid outlet 105.

Alternatively, the three-dimensional microelectrode array formed by the particle suspension inlet 101, the focusing sheath liquid inlet 102, the sample flow channel 103, the particle suspension outlet 104, and the focusing sheath liquid outlet 105, and the electrode flow channel 205 and the electrode flow channel 206, which are opposite in polarity, the liquid metal inlet 201 and the liquid metal inlet 203, and the liquid metal outlet 202 and the liquid metal outlet 204, may be fixed in an irreversible manner, or may be bonded in a reversible manner, so that the bio-particle separation device can be disassembled after processing one batch of samples, and re-bonded with a new substrate for the next batch of samples after necessary elution, sterilization, and the like.

Alternatively, the electrode runners may be connected together by wires to custom printed circuit boards that match their electrode patterns for the convenience of applying electrical signals. In an example, the signal generator may be connected via a power amplifier to an electrode runner filled with liquid metal, providing an electrical signal to the bio-particle separation device. Wherein the applied electrical signal can also be observed with an oscilloscope.

The biological particle separating device integrates the large-array three-dimensional liquid metal alloy microelectrode on the chip, is beneficial to constructing a high-flux dielectrophoresis microfluidic device and system, and realizes continuous, progressive and high-flux dielectrophoresis biological particle separation.

The working principle and structural design of the biological particle separating device according to the embodiments of the present disclosure are exemplarily described below.

The electrode runners (e.g., electrode runner 205 and electrode runner 206, which are of opposite polarity) have a laplace pressure for the incoming liquid metal therein that needs to be overcome to begin flowing the incoming liquid metal through the electrode runner. For example, if the surface of the incoming liquid metal in the electrode runner becomes higher, the surface of the incoming liquid metal bends during its ascent in the microchannel tube, creating a pressure differential that creates a laplace pressure that impedes the flow of the incoming liquid metal.

The micro-channels have different geometric shapes, so that the surface of the input liquid metal is bent differently, and the Laplace pressure can be determined by the geometric shapes of the micro-channels.

Fig. 3 is a schematic diagram illustrating the capillary valve principle according to an embodiment of the present disclosure, and as shown in fig. 3, if the shape of the electrode runner is a straight-flow channel as shown in part a of fig. 3, the cross-sectional shape thereof perpendicular to the flow direction of the liquid metal is rectangular, and the laplace pressure of the electrode runner can be calculated by the following formula:

in formula (1), ΔP _str The Laplace pressure of the electrode runner in the shape of a straight channel, gamma is the surface tension between liquid metal and gas, and for a solid-like film formed by the interfacial oxidation of the liquid metal and the gas, gamma depends on the mechanical property of the film, and theta _c The contact angle for the liquid metal to advance, w represents the width of the cross-sectional shape of the electrode runner perpendicular to the flow direction of the liquid metal, and h represents the height of the cross-sectional shape of the electrode runner perpendicular to the flow direction of the liquid metal.

Abrupt expansion of the electrode flowpath width or height may result in the occurrence of additional laplace pressure. As shown in part b of fig. 3, the laplace pressure of the electrode flow path having the expansion structure is as follows:

In formula (2), ΔP _exp For the Laplace pressure of the electrode runner with an expansion structure, gamma is the surface tension between liquid metal and gas, and for a solid-like film formed by the interfacial oxidation of the liquid metal and the gas, gamma depends on the mechanical property of the film, and theta _c Is the contact angle at which the liquid metal advances,beta is the cone angle at the expansion structure, w represents the width of the cross-sectional shape of the electrode runner perpendicular to the flow direction of the liquid metal, and h represents the height of the cross-sectional shape of the electrode runner perpendicular to the flow direction of the liquid metal.

As can be seen from the descriptions of the formula (1) and the formula (2), for the electrode flow channels having the same width w and height h, Δp _exp Greater than delta P _str . When the liquid metal reaches the junction of the straight flow channel and the expansion flow channel with the same width w and height h, the liquid metal can flow into the straight flow channel first due to the lower Laplacian pressure of the capillary valve corresponding to the straight flow channel. Therefore, by placing capillary valves with different trigger pressure thresholds at proper positions in the electrode flow channel, the flow direction of liquid metal in the electrode flow channel can be controlled, and the three-dimensional liquid metal electrode can be integrated to the greatest extent in the limited chip space of the biological particle separating device in the embodiment of the disclosure, so that the high-flux progressive dielectrophoresis sample treatment is realized.

In one possible implementation, the principle of implementing control of the flow of liquid metal by expanding the laplace pressure as a capillary valve by abrupt changes in the conduit dimensions in the electrode runner may be implemented by changing the electrode runner cross-sectional area (e.g., changing the width or height of the electrode runner).

In order to increase the spatial density of the liquid metal electrode in the bio-particle separation device of the embodiments of the present disclosure, the liquid metal may be provided with the ability to switch flow paths during filling of the electrode flow channels, thereby self-assembling a compact electrode pattern.

In one possible implementation, to reliably and efficiently achieve the goal of self-assembling liquid metal into a compact electrode pattern, an electrode runner may include capillary valves having different trigger pressure thresholds, and may provide robust passive liquid control capability using the capillary valves, and fig. 4 illustrates a schematic structural diagram of an electrode runner according to an embodiment of the present disclosure, and as illustrated in fig. 4, the capillary valve of the electrode runner may include a stop valve, a passive switching valve, through which the electrode runner communicates with the sample runner, and a first path of the electrode runner communicates with a branched second path through which the passive switching valve communicates, and a cross-sectional area S1 of the electrode runner is greater than a cross-sectional area S2 of the passive switching valve, and a cross-sectional area S2 of the passive switching valve is greater than a cross-sectional area S3 of the stop valve.

The first path refers to a trunk path where the current liquid metal flows currently, and the second path refers to a branch path. Taking fig. 1 as an example, the electrode runner 205 includes a first portion 205-1, a second portion 205-2 and a third portion 205-3, when the liquid metal enters the electrode runner 205 from the liquid metal inlet 203, the liquid metal is used as a first path, 205-2 is used as a second path, a passive switching valve is arranged at the intersection point of 205-1 and 205-2, so that the liquid metal is filled with 205-1, and then the filling of 205-2 is continued, at this time, 205-2 is used as the first path, 205-3 is used as the second path, and a passive switching valve is also arranged at the intersection point of 205-2 and 205-3, so that the filling of 205-3 is continued after the liquid metal is filled with 205-2, and finally the liquid metal outlet 204 is reached. Still referring to fig. 1 as an example, a plurality of shut-off valves may be arranged at specified intervals along the electrode flow path 205, and the embodiments of the present disclosure do not limit specific intervals and may be set according to specific application scenarios.

The electrode runner 206 is similar to the electrode runner 206, and for example, in fig. 1, the electrode runner 206 includes a first portion 206-1, a second portion 206-2, and a third portion 206-3, when the liquid metal enters the electrode runner 206 from the liquid metal inlet 201, the liquid metal is filled in the electrode runner 206 by taking the first portion 206-1 as a first path, the liquid metal is filled in the electrode runner 206 by taking the second portion 206-2 as a second path, and a passive switching valve is disposed at an intersection of the first portion 206-1 and the second portion 206-2, so that the liquid metal is filled in the electrode runner 206, and then the electrode runner 206 is filled in the liquid metal outlet 204 after the liquid metal is filled in the electrode runner 206 by taking the second portion 206-2 as a second path, and then the electrode runner 206-2 is continuously filled in the electrode runner 206-2. Still referring to fig. 1 as an example, a plurality of shut-off valves may be disposed at designated intervals along the electrode flow path 206, and the embodiments of the present disclosure do not limit specific intervals and may be set according to specific application scenarios.

The sectional area S1 of the electrode flow channel represents the area of a section perpendicular to the flow direction of the liquid metal in the electrode flow channel, the sectional area S2 of the passive switching valve represents the area of a section perpendicular to the flow direction of the liquid metal in the passive switching valve, and the sectional area S3 of the shut-off valve represents the area of a section perpendicular to the flow direction of the liquid metal in the shut-off valve.

As can be seen by combining equations (1) and (2), the size of the cross section is inversely related to the trigger pressure threshold that blocks the flow of the liquid metal, and the larger the area of the cross section is, the smaller the trigger pressure threshold is; the smaller the area of the cross section, the greater its corresponding trigger pressure threshold. Because the sectional area S1 of the electrode flow channel is larger than the sectional area S2 of the passive switching valve and larger than the sectional area S3 of the stop valve, the trigger pressure threshold value of the electrode flow channel is smaller than the trigger pressure threshold value of the passive switching valve and smaller than the trigger pressure threshold value of the stop valve.

In this arrangement, the shut-off valve is configured to prevent the liquid metal from entering the sample flow path after filling the first path of the electrode flow path; the passive switching valve is used for: and in case the first path of the electrode runner is not filled with liquid metal, preventing the liquid metal from entering the branched second path, and in case the first path of the electrode runner is filled with liquid metal, switching the flow direction of the liquid metal from the first path to the branched second path.

As shown in fig. 4, different constriction structures are suitably arranged in the electrode flow channels in the biological particle separation apparatus, which constriction structures act as capillary valves with specific trigger pressure thresholds, enabling passive and accurate flow path (e.g. first and second path) control of the liquid metal.

As an example, the electrode flow channel is connected to the sample flow channel by a square aperture (see S3 of fig. 4) with a side length of 10 microns, which aperture has the highest trigger pressure threshold, and can be used as a shut-off valve to prevent liquid metal from entering the sample flow channel after filling the first path. In addition, a constriction having a width of 80 μm may be placed at the branching of the electrode flow path as a passive switching valve (see S2 of fig. 4) whose trigger pressure threshold is stronger than that of the electrode flow path (see S1 of fig. 4) but weaker than that of the shut-off valve to switch to the second path of the electrode flow path after the liquid metal fills the first path of the electrode flow path along the first path of the electrode flow path.

In one possible implementation, the bio-particle separation device is fabricated by a soft lithography process, liquid metal can be injected into a liquid metal inlet of the bio-particle separation device, and the self-assembled electrode pattern of the bio-particle separation device is integrated into a large array of three-dimensional microelectrodes by using capillary valves with different trigger pressure thresholds. The self-assembly process of the biological particle separation device comprises the following steps: when the liquid metal is injected into the electrode runner and flows to the path branches, the passive switching valve blocks the liquid metal from entering the second paths of the branches, and the liquid metal flows forwards along the first paths; under the condition that the first path of the electrode runner is filled with the liquid metal, the stop valve prevents the liquid metal from entering the sample runner after the first path of the electrode runner is filled with the liquid metal, and the passive switching valve switches the flow direction of the liquid metal from the first path to the branched second path; and when the liquid metal enters the branched second path, taking the branched second path as the first path of the trunk, and repeatedly executing the process until the flowing liquid metal fills the electrode runner to form an electrode pattern.

It can be seen that the embodiments of the present disclosure can utilize the passive fluid control capability of the gradient threshold capillary valve, provide a simple and reliable liquid metal electrode structure and manufacturing method, realize self-assembly in a compact dielectrophoresis microfluidic device to form a three-dimensional liquid metal electrode array, so as to significantly improve the flux of the biological particle separation device, and fill the gap between the clinical sample processing requirements and the microfluidic biological particle separation technical flux.

Fig. 5 shows a schematic diagram of a liquid metal electrode array self-assembly workflow of an embodiment of the present disclosure, which illustrates an example of a path switching process performed by liquid metal in the process of filling an electrode flow channel under a capillary force as shown in formulas (1) and (2). Wherein the cross section of the current flow channel, the passive switching valve and the stop valve is rectangular, the heights of the current flow channel, the passive switching valve and the stop valve are the same, and the width W of the electrode flow channel ₁ Width W of passive switching valve ₂ Width W of > stop valve ₃ So the sectional area S1 of the electrode flow channel is larger than the sectional area S2 of the passive switching valve and larger than the sectional area S3 of the stop valve, thereby the electrode flow channel is contactedThe trigger pressure threshold value is smaller than the trigger pressure threshold value of the passive switching valve and smaller than the trigger pressure threshold value of the stop valve. It should be understood that the present disclosure takes only a rectangle as an example, and the shapes of the current flow path, the passive switching valve, and the shutoff valve are not particularly limited.

First, when liquid metal is injected into the electrode runner and flows to the path branches (at the junction of the first path and the second path in fig. 5), the liquid metal may selectively fill the electrode runner along the first path or the second path of the branches of the trunk. Because the trigger pressure threshold of the passive switching valve is higher than that of the electrode runner, the passive switching valve blocks the liquid metal from entering the second path of the branch, so that the liquid metal flows forwards along the first path.

The liquid metal fills the first path and reaches the capillary valve structure serving as a strong trigger pressure threshold of the stop valve, and the passive switching valve still prevents the liquid metal from entering the branched second path.

With the rise of the liquid metal flowing pressure, when the liquid metal flowing pressure reaches a weak trigger pressure threshold of the passive switching valve, the liquid metal can break through the passive switching valve and start to fill the second path of the branch because the trigger pressure threshold of the stop valve is higher than the trigger pressure threshold of the passive switching valve, and the stop valve prevents the liquid metal from entering the sample flow channel.

When the liquid metal enters the branched second path, the branched second path can be used as the first path of the trunk, the branched path on the path is used as the second path, the process is repeatedly executed, the flowing liquid metal can autonomously fill the electrode runner with a complex pattern, so that a compact and large-sized three-dimensional electrode array is formed, and meanwhile, the probability that the electrode runner is not filled and the liquid metal leaks into the sample runner is greatly reduced by the mechanism. According to this mechanism, each pair of electrodes can have the same structure and operating condition during autonomous filling of the electrode runners with liquid metal, so that the electrode array can be infinitely expanded.

Compared with the prior art, the electrode has a simple structure and the manufacturing method is not practical. The embodiment of the disclosure designs a large-array electrode connection structure and provides a capillary-based three-dimensional liquid metal alloy electrode self-assembly method. The complex three-dimensional liquid metal electrode pattern can be self-assembled by only manually pushing the injector to inject and drive, so that the reliability and the expandability of the manufacturing process are improved. In addition, the embodiment of the disclosure realizes the integration of a large array of three-dimensional liquid metal electrodes in a chip through the accurate control of the liquid metal flow, and further generates a strong and uniform three-dimensional electric field in the height direction of the whole sample flow channel. The large-scale three-dimensional electric field enables efficient particle manipulation, and provides a powerful technical means for separation and analysis of biological samples.

In one possible implementation, the electrode runner employs an asymmetric electrode arrangement, with each shut-off valve of the positive side arrangement corresponding to a plurality of shut-off valves of the negative side arrangement (e.g., including 3, 4, 5, etc.), or with each shut-off valve of the negative side arrangement corresponding to a plurality of shut-off valves of the positive side arrangement (e.g., including 3, 4, 5, etc.).

For example, as shown in fig. 2, along the sample flow channel 103, a first pair of three-dimensional microelectrodes 401 may be set to have one stop valve corresponding to N1 stop valves, a second pair of three-dimensional microelectrodes 401 may be set to have one stop valve corresponding to N2 stop valves, a third pair of three-dimensional microelectrodes 401 may have one stop valve corresponding to N3 stop valves, and values of N1, N2, and N3 may be the same or different, where the number of stop valves in each pair of three-dimensional microelectrodes is not limited in the embodiments of the disclosure.

In order to generate the non-uniform electric field required for dielectrophoresis in the sample flow channel, each pair of microelectrodes employs an asymmetric electrode arrangement. For example, a stop valve (for example, small holes) is arranged on the positive electrode side, and a plurality of stop valves (for example, a plurality of small holes) are correspondingly arranged on the negative electrode side; for another example, a single shut-off valve (e.g., small holes) is provided on the negative electrode side, and a plurality of shut-off valves (e.g., small holes) are provided on the positive electrode side. It should be understood that the size of the apertures may be selected as desired, e.g., 10 microns, and the present disclosure is not limited to the number and size of apertures.

Dielectrophoresis describes, in an example, the translational movement of neutral particles located in a non-uniform electric field due to the effect of dielectric polarization.

Fig. 6 shows a schematic diagram of a uniform sphere model and a single shell sphere model of an embodiment of the present disclosure, the principles of dielectrophoresis effect are described below in connection with fig. 6.

The particles in the biological fluid to be tested can be described based on a uniform sphere model, which is assumed to have dielectric properties as shown in part a on the left side of fig. 6. The time-averaged dielectrophoretic force to which uniform spherical particles suspended in a medium are subjected can be expressed as follows:

in formula (3), F _DEP Representing the time-averaged dielectrophoretic force to which uniform spherical particles suspended in a medium are subjected, R being the particle radius, ε _m Is the dielectric constant of the medium, E is the electric field strength,re [. Cndot.]Representing the real part, K, of the complex variable _CM The clausius-Mo Suodi coefficient for uniform spherical particles can be expressed as follows:

in the formula (4) of the present invention,for the complex permittivity of the particles as a function of frequency, < >>For the complex permittivity of the medium varying with frequency, the complex permittivity of the polarizable particles is defined by +.>The complex dielectric constant of the medium is obtained byObtained, wherein σ _p Representing the conductivity, sigma, of the particles _m Representing the conductivity of the medium. j is an imaginary unit, ω is the angular frequency of the ac signal.

While most biological cells, which can be considered to consist of a membrane forming a vesicle structure, their dielectric properties cannot be simplified by the uniform sphere model described above. These particles can be characterized by a single shell sphere model, as shown in part b on the right side of fig. 6. clausius-Mo Suodi coefficient K of single shell sphere particles _CM The method can be expressed as follows:

in equation (5), the effective complex permittivity of the film-coated particlesComplex dielectric constant of uniform spherical particles in the formula (4) is replaced by +>Complex dielectric constant of membranous particles->The method can be expressed as follows: />

In the formula (6) of the present invention,complex permittivity indicative of cytoplasm, +.>Representing the complex permittivity of the membrane, R representing the outer radius of the single shell dielectric model, d representing the membrane thickness of the single shell dielectric model, the complex permittivity of the cytoplasm +.> Complex dielectric constant of film->The complex area of the membrane is less than the membrane capacitance> Is the area ratio of the film to the film capacitance, +.>Is the area ratio of the membrane to the membrane conductivity. Sigma (sigma) _cyto Representing the conductivity, sigma, of the cytoplasm _mem Indicating the conductivity of the cell membrane.

Fig. 7 is a schematic diagram illustrating electric field intensity simulation of a three-dimensional microelectrode according to the embodiment of the present disclosure, as shown in fig. 7, in order to generate a non-uniform electric field required for dielectrophoresis in a sample flow channel, each pair of grid units (e.g., 401 in fig. 2) of the electrode flow channel shown in part a of fig. 7 is configured by using an asymmetric electrode, one small hole is disposed on the positive electrode side, a plurality of small holes are disposed on the negative electrode side, and the sizes of the small holes serving as stop valves can be selected according to the needs, e.g., 10 micrometers, which is not limited by the present disclosure. In addition, a small hole may be provided on the positive electrode side, and N (N > 1) small holes may be provided on the negative electrode side, for example, N may take values of 3, 4, 5, 6, etc., and the number of small holes on the negative electrode side is not particularly limited in the present disclosure. The simulation results of the electric field formed by the asymmetric electrode arrangement are shown in part B and part C of FIG. 7, wherein part B is a sectional view of the simulation results of the electric field intensity in the section A-A of the sample flow channel, and part C is a top view of the simulation results of the electric field intensity in the sample flow channel. In the biological particle separating apparatus, a plurality of pairs of three-dimensional microelectrodes shown in part A of FIG. 7 are arranged in sequence along the sample flow channel, and biological particles described in part D of FIG. 7 pass through simulation results of motion trajectories of the 1 st pair of three-dimensional microelectrodes (see part I of the drawing), the 15 th pair of three-dimensional microelectrodes (see part II of the drawing), the 30 th pair of three-dimensional microelectrodes (see part III of the drawing) and the 50 th pair of three-dimensional microelectrodes (see part IV of the drawing), respectively.

Simulation results show that the three-dimensional sidewall electrode formed from liquid metal introduces a non-uniform strong electric field throughout the depth of the sample flow channel. This design has significant advantages over planar electrode arrangements. Firstly, the configuration of the side wall electrode does not interfere with sample imaging, so that real-time observation is convenient to realize. Second, a higher separation flux can be achieved without the need to reduce the flow channel height to accommodate the range of action of the electrodes. Further, the high conductivity of the liquid metal can minimize circuit losses, allowing dielectrophoretic deflection at lower voltages.

To initially verify the protocol, a bio-particle separation device integrated with 50 pairs of three-dimensional electrodes was first tested to separate samples of sea-drag cells (HeLa cells) and 10 micron polystyrene microbeads (PS beads) at 9 μl/min.

Fig. 8 shows a schematic diagram of a validation experiment of a biological particle separation device of an embodiment of the disclosure. Wherein, part a of fig. 8 shows the trajectories of the mixed sample composed of the sea-tangled cells and the polystyrene microbeads at the 1 st counter electrode (see row i column in fig. 8), the 30 th counter electrode (see row ii column in fig. 8), and the exit (see row iii column in fig. 8) without dielectrophoresis force; part B of fig. 8 shows the trajectory of a mixed sample of sea-going cells and polystyrene microbeads with dielectrophoretic forces (e.g., dielectrophoretic forces generated by an electric field of 40V frequency 1 MHz), the 1 st pair of electrodes (see row i column B in fig. 8), the 30 th pair of electrodes (see row ii column B in fig. 8) and the exit (see row iii column B in fig. 8); part C of fig. 8 shows the separation efficiency of the sea-going cells and the polystyrene microbeads; part D of fig. 8 shows purity of the sea-tangle cells and polystyrene microbeads at the inlet and outlet.

As can be seen from the comparison experiment in FIG. 8, particle trajectories in continuous separation are shown with and without dielectrophoretic force signals, and sea-Law cells and polystyrene are calculatedSeparation efficiency and purity of microbeads. Wherein, with the biological particle separation device of the embodiment of the disclosure, about 99.31% of polystyrene microbeads deflect and go to O under the influence of dielectrophoresis force ₂ Move, whereas about 89.29% of the sea-Law cells move from O ₁ And (5) removing. At the same time, sea Law cells in O ₁ The purity of the polystyrene microsphere is up to 99.19%, and the polystyrene microsphere is prepared by the method that O is ₂ The purity of the product was 85.47%. The enrichment was significant compared to the initial ratio of 2:1 for both particles.

Therefore, according to the biological particle separating device disclosed by the embodiment of the disclosure, the separation of different types of particles can be efficiently and accurately realized according to the deflection difference of different particles under the action of dielectrophoresis force.

To further verify the effectiveness of the large array three-dimensional liquid metal microelectrode structure and the processing method proposed by the present disclosure, 10 micrometer diameter polystyrene microspheres are used as a sample to be tested, and a biological particle separation device integrated with 5000 pairs of three-dimensional microelectrodes is introduced at a flow rate of microliters/min, fig. 9 is a schematic diagram showing another verification experiment of the biological particle separation device according to an embodiment of the present disclosure, where part a of fig. 9 shows that the voltage is 20V _pp Particle trajectories of polystyrene microsphere samples passing through the first pair (i), fifth hundred pairs (ii) and fifth thousand pairs (iii) of electrodes at a frequency of 100 kHz; FIG. 9 part b shows probability density function E of particle trajectories of polystyrene microsphere samples passing through the first, fifth hundred and fifth thousand pairs of electrodes _index 。

As shown in part a (i) of fig. 9, when the sample passes through the first pair of three-dimensional microelectrodes, the sample particles rapidly pass through the effective electric field region due to the high sample flow velocity, and little deflection of the particle trajectories due to dielectrophoresis forces is observed. As shown in part a (ii) of fig. 9, the cumulative effect of dielectrophoretic deflection by the three-dimensional microelectrode array, when the sample passes through the fifth hundred pairs of three-dimensional microelectrodes, the particle trajectories exhibit a deflection of about 6.2 microns, but the deflection distance is small enough to achieve effective dielectrophoretic particle separation. Finally, as shown in part a (iii) of fig. 9, a significant deflection of the particle trajectory was observed when the sample passed through the fifth thousand pairs of three-dimensional microelectrodes, a deflection distance of about 40 microns, which was 6 times greater than when the sample passed through the fifth hundred pairs of three-dimensional microelectrodes, sufficient to affect the exit of the sample flow direction.

In the above-described validation experiment integrating 5000 pairs of microelectrode device examples, an effective dielectrophoretic deflection of about 40 microns was achieved at a sample flow rate of microliters/minute, greatly improving the sample separation throughput.

In summary, in the biological particle separating device according to the embodiment of the present disclosure, the electrode flow channel and the sample flow channel are designed together, and capillary valves with different trigger pressure thresholds are disposed in the electrode flow channel, so as to realize path connection and switching of the electrode flow channel, thereby manufacturing a three-dimensional microelectrode pair array self-aligned with the sample flow channel; the biological particle separating device disclosed by the embodiment of the disclosure realizes accurate control of liquid metal flow by utilizing capillary action, and can be self-assembled into a complex electrode pattern by driving the liquid metal to flow by a hand-push injector, so that the integration of high-density large-array three-dimensional liquid metal electrodes in a microfluidic chip is realized. By utilizing the biological particle separation device integrated with the large-array three-dimensional liquid metal electrode, high-flux dielectrophoresis microfluidic separation can be carried out on a biological particle sample to be detected, and accumulation and superposition of dielectrophoresis force effect are realized by integrating the large-array three-dimensional electrode in the microfluidic chip, so that high-flux progressive particle separation is realized.

The related art dielectrophoresis microfluidic method is limited by its low separation efficiency and flux at high sample flow rates and high sample concentrations. The disclosed embodiments enable continuous, progressive and high flux bio-particle separation by integrating large arrays of three-dimensional liquid metal electrodes. The effect of dielectrophoresis force is accumulated along with the number of electrode pairs by a large number of electric field gradient fields generated in the micro-flow channel by the large array electrodes, and sample particles can realize progressive deflection under high flow velocity and high concentration, so that the separation flux is remarkably improved.

In summary, the technical scheme of the invention provides a large-array three-dimensional liquid metal microelectrode structure and a manufacturing method thereof, improves separation flux, realizes efficient particle manipulation, overcomes the limitation of the prior art, brings remarkable beneficial technical effects, improves the number of arrays by at least 10 times, and improves the separation flux by 10 times. These effects make the present invention an innovative technology with important application potential and commercial value.

It will be appreciated by those skilled in the art that in the above-described method of the specific embodiments, the written order of steps is not meant to imply a strict order of execution but rather should be construed according to the function and possibly inherent logic of the steps.

The foregoing description of the embodiments of the present disclosure has been presented for purposes of illustration and description, and is not intended to be exhaustive or limited to the embodiments disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the various embodiments described. The terminology used herein was chosen in order to best explain the principles of the embodiments, the practical application, or the improvement of technology in the marketplace, or to enable others of ordinary skill in the art to understand the embodiments disclosed herein.

Furthermore, the terms "first," "second," and the like, are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include at least one such feature. In the description of the present invention, the meaning of "plurality" means at least two, for example, two, three, etc., unless specifically defined otherwise.

In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.

While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.

Claims

1. A biological particle separation device, the device comprising:

a microchannel comprising a particle suspension inlet, a focusing sheath liquid inlet, a sample channel, a particle suspension outlet, a focusing sheath liquid outlet, the particle suspension inlet and the focusing sheath liquid inlet communicating the particle suspension outlet and the focusing sheath liquid outlet via the sample channel;

the liquid metal inlet is communicated with the liquid metal outlet through the electrode runner, and the electrode runner is used for forming at least one group of three-dimensional microelectrodes with opposite polarities;

and the substrate is used for fixing the micro-flow channel, the liquid metal inlet, the electrode flow channel and the liquid metal outlet.

2. The apparatus of claim 1, wherein the electrode runner includes capillary valves having different trigger pressure thresholds, the capillary valves being used to control the flow direction of liquid metal in the electrode runner.

3. The device of claim 2, wherein the capillary valve comprises a shut-off valve, a passive switching valve, the electrode flow channel being in communication with the sample flow channel through the shut-off valve, a first path of the electrode flow channel being in communication with a branched second path through the passive switching valve, a cross-sectional area of the electrode flow channel being greater than a cross-sectional area of the passive switching valve, the cross-sectional area of the passive switching valve being greater than a cross-sectional area of the shut-off valve.

4. A device according to claim 3, wherein the shut-off valve is adapted to prevent the liquid metal from entering the sample flow path after filling the first path of the electrode flow path;

the passive switching valve is used for: preventing the liquid metal from entering the second path of the branch in the case of the liquid metal not filling the first path of the electrode runner, and

in case the first path of the electrode runner is filled with liquid metal, the flow direction of the liquid metal is switched from the first path to the branched second path.

5. The device of any one of claims 2-4, wherein the electrode flow channels are provided with asymmetric electrodes, each shut-off valve provided on the positive side corresponding to a plurality of shut-off valves provided on the negative side, or each shut-off valve provided on the negative side corresponding to a plurality of shut-off valves provided on the positive side.

6. The device according to any one of claims 1-4, wherein the particle suspension inlet and the particle suspension outlet are connected to a microfluidic pump by means of a plastic hose for controlling the flow of the particle suspension in the microfluidic channel by means of the microfluidic pump.

7. The apparatus of any one of claims 1-4, wherein the liquid metal comprises at least one of indium, tin, cadmium, bismuth, lead, gallium.

8. A method of processing a biological particle separation device, the method comprising:

manufacturing the bio-particle separation device according to any one of claims 1 to 7 by a soft lithography process;

and injecting liquid metal into a liquid metal inlet of the biological particle separating device, and integrating the electrode pattern self-assembled by the biological particle separating device into a large-array three-dimensional microelectrode by utilizing capillary valves with different trigger pressure thresholds.

9. The method of claim 8, wherein the capillary valve comprises a shut-off valve, a passive switching valve, the electrode flow channel being in communication with the sample flow channel through the shut-off valve, a first path of the electrode flow channel being in communication with a branched second path through the passive switching valve, a cross-sectional area of the electrode flow channel being greater than a cross-sectional area of the passive switching valve, the cross-sectional area of the passive switching valve being greater than a cross-sectional area of the shut-off valve;

The self-assembly process of the biological particle separation device comprises the following steps:

when the liquid metal is injected into the electrode runner and flows to the path branches, the passive switching valve blocks the liquid metal from entering the second paths of the branches, and the liquid metal flows forwards along the first paths;

the stop valve prevents liquid metal from entering the sample runner under the condition that the liquid metal fills the first path of the electrode runner, and the passive switching valve switches the flow direction of the liquid metal from the first path to the branched second path;

and when the liquid metal enters the branched second path, taking the branched second path as the first path of the trunk, and repeatedly executing the process until the flowing liquid metal fills the electrode runner to form an electrode pattern.

10. A microfluidic chip comprising the biological particle separation device of any one of claims 1-7.