CN115400879A - Microfluidic dielectrophoresis chip, manufacturing method thereof and application of chip in liquid metal particle sorting - Google Patents

Abstract

The present invention relates to a microfluidic dielectrophoresis chip, its manufacturing method and its application in the sorting of liquid metal particles. The microfluidic dielectrophoretic chip includes a microelectrode and a microchannel. The microelectrode includes a glass substrate and Two metal chromium electrodes on the metal chromium electrode, each metal chromium electrode includes a wave section and a pin section connected to the end of the wave section, the wave sections of the two metal chromium electrodes are parallel to each other, and the leads of the two metal chromium electrodes The foot sections are away from each other; the microchannel is a PMDS cover sheet with a concave channel on the lower surface, the microchannel is bonded and packaged with the microelectrode through PMDS, and the wave sections of the two metal chromium electrodes are located in the concave channel of the microchannel . The invention realizes particle sorting and capturing functions for liquid metal particles through dielectrophoretic microfluidic technology, and its sorting efficiency reaches over 95%, which can broaden the application of dielectrophoretic microfluidic technology in biomarker detection and cell sorting .

Description

A microfluidic dielectrophoresis chip and its manufacturing method and sorting of liquid metal particles application in

technical field

The invention relates to the field of sorting chips, in particular to a microfluidic dielectrophoresis chip, a manufacturing method thereof and an application in sorting liquid metal particles.

Background technique

In recent decades, microfluidic technology has attracted extensive attention due to its advantages of miniaturization, portability, integration, automation, low cost, high throughput, and simple operation. It is an interdisciplinary field that integrates the perspectives of chemistry, physics, life sciences, microelectronics, materials science, computer science, etc., and has been applied in in vitro diagnostics (IVD), liquid biopsy, environmental and biochemical analysis, single cell analysis, Nucleic acid analysis, etc. More specifically, routine analysis and analysis steps such as mixing, separation, enrichment, manipulation, sorting detection, synthesis, and cell culture are realized in small chips and microfluidic systems. The ability of microfluidics to separate cells quickly and efficiently often relies on external force fields, such as optics, electronics, magnetic fields, or acoustics. Acoustofluidic and radiative force-based BAW manipulation holds great promise for a wide range of applications, with advantages including versatility, biocompatibility, precision, flexibility, compactness, and cost-effectiveness, as well as ease of integration with other microfluidic technologies. To date, a wide range of particles ranging in size from millimeter to millimeter in various fluid media such as air, whole blood or sputum have been successfully controlled using this technique.

The development status of microfluidic technology; microfluidic dielectrophoresis technology has developed very rapidly in recent years. In 1999, Morgan et al. designed a polynomial electrode to realize the detection of tobacco mosaic virus (TMV) and herpes simplex virus (HSV) Ⅰ Type capture and classification; Li et al. proposed a wave-shaped microfluidic dielectrophoresis chip in 2012, which achieved the separation of polystyrene particles and yeast cells under different DC voltages; Pilloni et al. A new design was proposed in 2016 to manipulate particles in three dimensions via dielectrophoretic forces using individually addressable planar and three-dimensional carbon microelectrodes.

L.Yu, C.Iliescu and others designed the discontinuous flow separation chip model. The shape and size of the microchannel of this model can change, resulting in changes in the distribution of the electric field in the channel, forming a non-uniform electric field. The working process of the chip is as follows: two mixed samples of A and B are passed into the microchannel. Due to the effect of non-uniform electric field, the dielectric responses of different samples are different, causing the samples to gather in different positions in the channel. At this time, when pressure is applied to one end of the channel, the A particles gathered in the middle of the channel will flow with the air pressure and flow out of the channel first. After all the A particles in the channel flow out, the electrical signal in the channel is disconnected, and the B particles in the groove will eventually flow out with the airflow because there is no dielectrophoretic force, so that the separation of the two mixed samples can be realized in time. In order to realize the separation operation of particles of different sizes, Yusukawa et al. improved the interdigitated electrodes in 2009. The design concept of this structure is relatively novel. Through the relationship between the dielectrophoretic force and the cube of the particle diameter, a microelectrode is designed. When particles of different sizes pass through the channel, they are gradually distributed in the electrode structure due to the effect of different forces. at different locations. The picture on the right shows that when the mixed solution of two particles with a diameter of 10 μm and 3 μm is injected into the channel, the two particles are subjected to negative dielectrophoretic force, so they are mixed together and pass through the flow channel. After a period of time, it can be seen in the outlet area that the gap between the two narrow electrodes There are only particles with a diameter of 3 μm between them, while particles with a diameter of 10 μm are between the two electrodes, one wide and one narrow. This is because particles with larger diameters in the channel are gradually transferred to the designated area under the action of a strong electric field at the wide electrode. The study of separation by dielectrophoresis has entered a period of rapid development since the 1990s. Many research groups in the world have realized the operation of particle separation by dielectrophoresis. For example, Morgan et al. have carried out the separation of polystyrene microspheres of different sizes. Separation, Yang and others carried out differential analysis of white blood cells; domestic Tsinghua University, Southeast University and other units have also carried out in-depth research in this field.

Inspired by the new concept of a three-dimensional fluid control platform based on cell structure, Yang Chaoyu et al. proposed the viewpoint of tissue engineering based on cell fluid vascular network construction. Red blood cells [diameter 6-8μm], one of the key indicators in clinical medicine, are isolated from whole blood and are of great practical importance since many biological targets span the same size range. For example, Huang et al. isolated exosomes from whole blood with a high blood cell removal rate by integrating a high-frequency (39.4 MHz) interdigital transducer (IDT). They demonstrated an acoustofluidic platform that can encode droplets via (FIDTs) with an optimal driving frequency of 96.25 MHz. Lee et al. optimized the design of high-frequency (394 MHz) IDT and underlying electronics to isolate nanoscale and microscale vesicles from cell culture media with high isolation yield and resolution. Additionally, Huang et al. designed A simple, low-cast nature and open fluid chamber platform utilizing phase modulation between two low-frequency [4-6kHz] piezoelectric transducers to achieve dynamic particle concentration and particle vortex translation with only one side Operate the vortex in a single direction.

The inhalation of heavy metals caused by daily eating habits or environmental factors or the accumulation of heavy metals caused by food will cause the blood of the human body to contain metal particles, and the accumulation of metal ions in the blood of the human body will cause chronic poisoning of the human body. leading cause of disease and premature aging. At the same time, in many fields, from food and water safety to environmental monitoring and clinical analysis, it is closely related to human health. Therefore, purifying such harmful particles as metal particles in blood or drinking water can prevent and treat diseases and is harmful to human health. has important meaning. The metal particles stagnant in human blood include liquid metal particles. At present, no one has used microfluidic technology to manipulate liquid metal particles to achieve their separation in blood.

Contents of the invention

The present invention aims to provide a microfluidic dielectrophoresis chip, its manufacturing method and its application in the sorting of liquid metal particles, so as to realize particle sorting and capture functions for liquid metal particles through dielectrophoretic microfluidic technology.

In order to solve the above technical problems, the specific solution adopted by the present invention is: a microfluidic dielectrophoresis chip, including a microelectrode and a microchannel, the microelectrode includes a glass substrate and two metal chromium electrodes on the glass substrate, each Each metal chromium electrode comprises a wave segment and a lead segment connected to the end of the wave segment, the wave segments of the two metal chromium electrodes are parallel to each other, and the lead segments of the two metal chromium electrodes deviate from each other; the microchannel A PMDS cover sheet with a concave channel is provided on the lower surface, the microchannel is bonded and packaged with the microelectrode through the PMDS, and the wave sections of the two metal chromium electrodes are located in the concave channel of the microchannel.

As a further optimization of the above technical solution, the width of the wavy section is 100 μm, the distance between the wavy sections of the two metal chromium electrodes is 100 μm, and the width of the sorting area formed by the two wavy sections is 850 μm.

As a further optimization of the above technical solution, the microchannel includes a channel body, two inlets at one end of the channel body and an outlet at the other end of the channel body, the channel body includes an inlet section, a sorting section and an outlet section connected in sequence, One end of the selection segment communicates with two inlets through two inlet segments, and the other end of the sorting segment communicates with the outlet through an outlet segment.

As a further optimization of the above technical solution, the depth of the concave channel is 0.5 mm, the width of the sorting section is 2 mm, and the widths of the entrance section and the exit section are both 1 mm.

A method for making a microfluidic dielectrophoresis chip, comprising the following steps

S1: Fabrication of microelectrodes: Metal chromium electrodes are fabricated on glass substrates by photolithography and wet etching to form microelectrodes;

S2: Fabricate the microchannel: take another glass substrate, enclose a housing cavity on the glass substrate, fix the microchannel male mold in the housing cavity, pour PDMS into the housing cavity and perform curing treatment to form a PMDS cover sheet; After taking out the PMDS cover sheet from the holding chamber, take out the male mold to form a microchannel, wherein the position of the male mold is a concave channel;

S3: Micro-electrode and micro-channel bonding, packaging: apply PMDS to the lower surface of the PMDS cover sheet, and cover it on the glass substrate where the metal chromium electrode is located, so that the wave section of the metal chromium electrode is located in the concave channel, after curing treatment , that is, the microfluidic dielectrophoresis chip was prepared.

As a further optimization of the above technical solution, in step S2, the accommodating chamber is surrounded by a glass substrate and four glass slides, and the four slide glass are connected end to end and bonded to the glass substrate.

As a further optimization of the above technical solution, in step S2, the male mold includes a slab and protrusions fixed at both ends of the slab for forming the inlet and outlet of the microchannel respectively.

As a further optimization of the above technical solution, in step S2, the height of the PDMS poured into the accommodating cavity is not higher than the height of the protrusion.

As a further optimization of the above technical solution, in step S2, the curing treatment specifically includes: heating at an ambient temperature of 80° C. for 30 minutes.

An application of a microfluidic dielectrophoresis chip in the sorting of liquid metal particles. First, a solution to be sorted containing liquid metal particles is passed into the concave channel from an inlet of the microchannel, and the solution to be sorted is filled with the concave channel. , turn on the ultrasonic power amplifier connected to the microfluidic dielectrophoresis chip and the function signal generator connected to the ultrasonic power amplifier, adjust the parameters of the function signal generator and provide non-uniform Electric field, the liquid metal particles are captured by the metal chromium electrode under the action of a non-uniform electric field, and then close the entrance of the solution to be sorted, and pass into the medium solution from the other entrance of the microchannel, and disconnect the ultrasonic power amplifier at the same time, the liquid metal After losing the capture of the metal chromium electrode, the particles flow out of the concave channel with the medium solution to achieve sorting.

Compared with the prior art, the beneficial effects of the present invention are as follows: the present invention realizes the functions of sorting and capturing liquid metal particles through dielectrophoretic microfluidic technology, and its sorting efficiency reaches over 95%, which can broaden the range of dielectrophoretic microfluidic control. Application of technology in cell sorting and biomedical engineering, medical devices and other fields.

Description of drawings

Fig. 1 is a structural schematic diagram of a microfluidic dielectrophoresis chip;

Figure 2 is an enlarged view of the microelectrode;

Fig. 3 is the production flowchart of microelectrode;

Fig. 4 is the overall structure schematic diagram of microelectrode;

Fig. 5 is a microchannel size diagram;

Fig. 6 is a structural schematic diagram of the accommodation cavity;

Fig. 7 is the structural representation of male mold;

Fig. 8 is a schematic diagram after the male mold is fixed on the accommodating cavity;

Fig. 9 is a schematic diagram of the stress of the particles in the microchannel during the sorting of the EGaln-red blood cell mixed solution;

Figure 10 is a schematic diagram of the distribution of eutectic gallium indium and red blood cells before power-on during the sorting of the EGaln-red blood cell mixed solution;

Figure 11 is a schematic diagram of the distribution of eutectic gallium indium after power is applied during the sorting of EGaln-red blood cell mixed solution;

Figure 12 is a schematic diagram of the distribution of red blood cells after power-on during the sorting of the EGaln-red blood cell mixed solution;

Figure 13 is the distribution diagram of the proportion of eutectic gallium indium and red blood cells in the solution before and after power-on when the ImageJ software sorts the EGaln-red blood cell mixed solution;

Figure 14 is a physical picture of the sorting effect of single eutectic gallium indium particles;

Figure 15 is a mixed solution sample of eutectic gallium indium and red blood cells;

Fig. 16 is a schematic diagram of a sorting operating system equipped with a microfluidic dielectrophoresis chip.

Detailed ways

Example 1

As shown in Figure 1, Figure 2 and Figure 4, the present embodiment is a microfluidic dielectrophoresis chip, including a microelectrode and a microchannel, and the microelectrode includes a glass substrate and two metal chromium electrodes positioned on the glass substrate .

The glass substrate is made of quartz, and each metal chromium electrode includes a wave section and a lead section connected to the end of the wave section. The wave sections of the two metal chromium electrodes are parallel to each other, and the lead sections of the two metal chromium electrodes are connected to each other. Deviation; the width of the wave section is 100 μm, the distance between the wave sections of the two metal chromium electrodes is 100 μm, and the width of the sorting area formed by the two wave sections is 850 μm. In this embodiment, the lead segments of the two metal chromium electrodes are parallel to each other and located on both sides in the width direction of the sorting area, and the thickness of the chromium layer of the metal chromium electrodes is about 100 nanometers.

Because the wavy shape of the wavy section can also be called "W-shaped", the metal chromium electrode with the wavy section in this embodiment is also called "W-shaped electrode". Compared with point electrodes and straight electrodes, W-shaped electrodes have higher capture efficiency higher.

As shown in Fig. 1, Fig. 5, described microchannel is that the lower surface is provided with the PMDS cover sheet of concave channel, microchannel is bonded and encapsulated by PMDS and microelectrode, and the wave section of two metal chromium electrodes is all positioned at the microchannel Inside the concave channel. The wave section has the same length direction as the concave channel and is located at the center of the concave channel.

Described micro channel comprises channel body, two inlets positioned at one end of channel body and an outlet positioned at the other end of channel body, channel body comprises the entrance segment, sorting segment and outlet segment that are connected successively, and one end of sorting segment passes through two inlets respectively The segment communicates with two inlets, and the other end of the sorting segment communicates with the outlet through an outlet segment. The depth of the concave channel is 0.5mm, the width of the sorting section is 2mm, the width of the entrance section and the exit section are both 1mm, and the total length of the concave channel is 35mm.

The two inlet sections are the upper inlet section and the lower inlet section respectively, the inlet end of the upper inlet section is set as the upper inlet, the inlet end of the lower inlet section is set as the lower inlet, and the outlet end of the upper inlet section and the outlet end of the lower inlet section meet Afterwards, it is connected with the sorting section, wherein, the lower inlet section, the sorting section and the outlet section are coaxially distributed, and the angle between the upper inlet section and the lower inlet section is an acute angle.

Example 2

This embodiment is the fabrication method of the microfluidic dielectrophoresis chip described in embodiment 1, comprising the following steps

S1: Fabrication of microelectrodes: Metal chromium electrodes are fabricated on glass substrates by photolithography and wet etching to form microelectrodes;

Specifically, the process flow is shown in Figure 3, and the manufacturing method is mainly divided into the following steps:

a. Cleaning: First, clean the glass substrate in an ultrasonic cleaning machine carrying deionized water. The glass substrate is made of quartz. After cleaning with deionized water, place the quartz glass substrate in the prepared acid solution for pickling . After the pickling is completed, it is finally cleaned with deionized water. The cleaning work seems simple, but it is not. If it is not cleaned, the produced microelectrodes will contain impurities and affect the results.

b. Electroplating: Fix the cleaned quartz glass substrate in the electroplating tank of the electroplating studio, set the parameters of the electroplating gun, and electroplate a chromium layer of about 100 nanometers.

c. SU-8 photoresist spin coating: place the quartz glass substrate after the chrome plating layer on the spin coating platform, use a spin coater to spin coat a layer of SU-8 photoresist, and the SU-8 photoresist after spin coating The resist is evenly distributed on the surface of the glass substrate. Put the spin-coated quartz glass substrate in a vacuum drying oven at 80°C and heat it for 30 minutes, and wait for the solvent to evaporate, so as to increase the bonding of the SU-8 photoresist to the glass substrate.

d. Exposure: Exposure is the most important link here. If the exposure is not good, it will affect the production accuracy of the microelectrodes. Take the spin-coated quartz glass substrate into the ultraviolet exposure machine, turn on the switch of the ultraviolet exposure machine, set the exposure time to 5 seconds, and perform exposure.

e. Developing: Place the exposed quartz glass substrate in the configured developing device for developing to remove unnecessary photoresist.

f. Wet etching: After the excess photoresist is removed, wet etching is required to leave the required electrode chromium layer and not corrode the chromium layer. Put the developed quartz glass substrate in the prepared acidic solution to etch for 3 minutes.

g. Cleaning: The quartz glass substrate after wet etching needs to be cleaned with deionized water.

h. Glue removal: This operation is to remove the glue on the electrode and expose the electrode.

i. Cleaning: clean the chromium electrode.

k. Laser cutting and packaging: At this point, the production of metal chromium electrodes is completely completed, and the quartz glass substrate is cut to the required size, and then packaged to prevent dust from falling. The obtained metallic chromium electrode is shown in Fig. 4 .

S2: Fabricate the microchannel: take another glass substrate, enclose a housing cavity on the glass substrate, fix the microchannel male mold in the housing cavity, pour PDMS into the housing cavity and perform curing treatment to form a PMDS cover sheet; After the PMDS cover sheet is taken out from the accommodating cavity, the male mold is taken out to form a microchannel, wherein the position of the male mold is a concave channel.

Microchannels need to be involved before making microchannels. Combined with the design of microelectrodes, the depth of concave channels in microchannels should not be too deep, otherwise the particles will be suspended in the solution, and the dielectrophoretic force will be very small if they are too far away from the non-uniform electric field; The width of the microchannel cannot be too wide, otherwise a large amount of particles will not flow over the microelectrode, but flow along the channel wall from both ends of the microelectrode. The designed channel size is shown in Figure 5. The depth of the concave channel is 0.5 mm, the width of the sorting section is 2 mm, the width of the entrance section and the exit section are both 1 mm, and the total length of the concave channel is 35 mm.

Specifically include the following steps:

1) Fabrication of the holding chamber: use four Chuanfan brand glass slides, the size of the slides is 25mm×75mm, first fix one slide glass on the base of a large glass sheet with 502 glue, and then use 502 glue to make it The four glass slides are connected end to end and fixed on the base of the large glass slide. After the fixation is completed, there is a square cavity in the middle as a storage cavity for making microchannels. The square cavity is shown in Figure 6. What needs to be explained Yes, the accommodating cavity can be a cavity of other shapes besides a square cavity.

2) Manufacture of the male mold: In order to save costs and control the thickness of the microchannel, a 0.5mm thick acrylic plate is used to make the male mold. First use AutoCAD2020 version software to draw the graphics of the male mold, and output the drawn picture as a .wmf file, then use CorelLASER software to open the file, set the cutting speed to 2mm/s, the number of cutting times to 3 times, and the current parameter to 8A. After the parameter setting is completed, place the 0.5mm acrylic plate on the cutting platform of the laser engraving machine, start cutting, and wait for the cutting to be completed. The finally made positive mold is shown in Figure 7.

3) Fabrication of the microchannel: first, use deionized water to clean the male mold and the square cavity. After cleaning, set three protrusions on the male mold. Specifically, three small cylinders are fixed to the male mold by double-sided adhesive tape. On the inlet and outlet, use 502 glue to fix the male mold with a cylinder in the center of the square cavity.

After the positive mold is fixed, start to configure PDMS. Cut off half of the disposable paper cup and put it on the analytical balance. After peeling, add PDMS until the analytical balance shows a weight of 10g, then add 1g of curing agent, and put the prepared PDMS After uniform mixing, pour it into a square cavity, and the height of pouring PDMS in the cavity is not higher than the height of the protrusion. After standing for a period of time, put it in a vacuum drying oven and vacuumize for 30 minutes. After vacuuming, put it in a microwave oven at 80°C for 30 minutes to promote the curing of PDMS. After the PDMS is heated and cured, take it out of the PMDS cover sheet square cavity Take out the male mold again, wherein the position of the male mold is a concave channel, and the positions of the protrusions respectively form the entrance and exit of the concave channel, that is, the entrance and exit of the microchannel, thereby completing the making of the microchannel. The microchannel when the male mold is not taken out is shown in Figure 8.

S3: Microelectrode and microchannel bonding and packaging: apply a layer of PMDS on the lower surface of the PMDS cover sheet, and quickly cover the quartz glass substrate where the metal chromium electrode is located, so that the wave section of the metal chromium electrode is located in the concave channel , and then heated in a microwave oven at 80° C. for 30 minutes for curing treatment, that is, a microfluidic dielectrophoresis chip is prepared. The microfluidic dielectrophoresis chip bonded with PDMS is firm and does not leak the solution. The chip after bonding and packaging is shown in Figure 1.

Example 3

This embodiment is the application of the above-mentioned microfluidic dielectrophoresis chip in the sorting of liquid metal particles. The microfluidic dielectrophoresis chip particle sorting system can be used to manipulate liquid metal particles to separate liquid metal particles from whole blood, and can also separate liquid metal particles.

When the microfluidic dielectrophoresis chip is used in the sorting process of liquid metal particles, the microfluidic dielectrophoresis chip needs to be set in the sorting operating system, which also includes a signal delivery device, a particle delivery device, and a data acquisition device. .

The signal transmission device includes a function signal generator and an ultrasonic power amplifier, and provides signals of specific frequency, specific waveform and specific voltage for the microfluidic dielectrophoresis chip. The frequency, peak-to-peak value, waveform, and phase are set in advance, and the function signal generator sends out the set signal, which is amplified by the ultrasonic power amplifier to provide a non-uniform electric field for the microfluidic dielectrophoresis chip.

The particle delivery device includes a micro-syringe pump to provide particles for the microfluidic dielectrophoresis chip. In order to control the velocity of particles in the microfluidic dielectrophoresis chip, a micro-injection pump is selected. The micro-injection pump can not only meet the requirements of particle movement speed, but also control the movement of the syringe bidirectionally (ie, forward advancement and reverse extraction).

The microfluidic dielectrophoresis chip is the core of the entire sorting operating system and the key to particle sorting. The chip generates a non-uniform electric field after receiving the signal amplified by the ultrasonic power amplifier, and the particles are polarized in the non-uniform electric field to generate dielectric force, which will be offset under the action of the dielectric force, thereby realizing the separation of particles.

The data acquisition device includes a microscope and a computer, which are used to observe and record the operation phenomenon. The Leica microscope imported from Germany is selected and connected to the microscope through the computer. Choose the situation.

The application process of the microfluidic dielectrophoresis chip in the sorting of liquid metal particles is as follows:

Firstly, the solution to be sorted containing liquid metal particles is passed into the concave channel from one inlet of the microchannel, specifically, the lower inlet of the microchannel is kept closed, and the solution to be sorted containing liquid metal particles is passed from the upper inlet;

After the sorting solution is filled with the concave channel, turn on the ultrasonic power amplifier connected to the microfluidic dielectrophoresis chip and the function signal generator connected to the ultrasonic power amplifier, adjust the parameters of the function signal generator and amplify it through the ultrasonic power amplifier. The microfluidic dielectrophoresis chip provides a non-uniform electric field, and the liquid metal particles are captured by the metal chromium electrode under the action of the non-uniform electric field;

Then close the inlet of the solution to be sorted, and feed the medium solution from another inlet of the microchannel, that is, close the upper inlet, open the lower inlet and feed the medium solution from the lower inlet, and disconnect the ultrasonic power amplifier at the same time, the liquid metal particles will lose After the capture of the metal chromium electrode, the medium solution flows out of the concave channel to realize the separation.

Eutectic gallium indium particles (EGaln) are one of the liquid metal particles. Therefore, taking the eutectic gallium indium particles as an example, the sample solution (that is, the solution to be sorted) is sorted through the microfluidic dielectrophoresis chip. The sample solution of the liquid metal particles may be a sample solution containing only eutectic gallium indium particles, or a mixed sample solution containing eutectic gallium indium particles and red blood cells.

1. The continuous separation operation process of single eutectic gallium indium particles by dielectrophoresis chips is as follows:

1) Take out the packaged microfluidic dielectrophoresis chip (referred to as the dielectrophoresis chip), check whether the dielectrophoresis chip is damaged, and use an electric soldering iron to solder the two copper wires to the two pins of the dielectrophoresis chip respectively , Finally, use a multimeter to check whether the welding is qualified, and the dielectrophoretic chip can be used only after passing the test.

2) Turn on the power of the operating system, and check whether the instruments in the operating system can work normally. After the inspection is correct, stick a layer of double-sided adhesive around the bottom of the welded DEP chip to fix the DEP chip under the microscope. Adjust the microscope light source to transmitted light, and the magnification is five times. To adjust the focal length, first coarsely adjust the focus to find the sorting area of the dielectrophoretic chip, then finely adjust the focus to clearly see the sorting area of the dielectrophoretic chip on the computer monitor, and finally adjust the light intensity to avoid the light brightness being too high or too low. Thus affecting the shooting quality. It should be noted that the dielectrophoretic chip sorting area refers to the area where the concave channel of the microchannel and the wave section of the metal chromium electrode are located.

3) Connect the 5ml syringe to the infusion tube, then shake the eutectic gallium indium particle bead sample solution well, and quickly draw the syringe in reverse, so that the eutectic gallium indium particle bead is in the infusion tube, and put the infusion tube vertically Lifting, the end of the infusion tube away from the syringe is connected to the inlet of the micro-injection pump, and the outlet of the micro-injection pump is connected to the upper inlet of the dielectrophoresis chip through a rubber hose, that is, the upper inlet is connected to the eutectic gallium indium particle suspension. Take another 5ml syringe, draw 5ml of the medium solution, and connect the syringe to the inlet of another microinjection pump through the infusion tube, and the outlet of the microinjection pump is connected to the lower inlet of the dielectrophoresis chip through a rubber hose, that is, the lower The inlet is connected with the medium solution. It is worth noting here that because the eutectic gallium indium suspension precipitates too fast, usually after mixing and shaking for 1 minute, most of the eutectic gallium indium particle pellets have already precipitated, so the eutectic gallium indium particle cannot be directly reduced to a small size. The ball sample solution is placed in the syringe, otherwise the eutectic gallium indium particle pellets will precipitate, and the eutectic gallium indium pellets will not be transported to the dielectrophoretic chip, and the continuous sorting cannot be completed. Therefore, it is chosen to store the sample solution of the eutectic gallium indium microspheres in the infusion tube, and the microspheres are transported through the infusion tube, so as to reduce the influence caused by the precipitation of the eutectic gallium indium microspheres.

4) After the particle delivery device is connected, set the flow rate or flow rate, turn on the micro-injection pump for transporting the sample solution, turn off the micro-injection pump for transporting the medium solution, and slowly push the syringe connected to the infusion tube of the sample solution to make the eutectic gallium The indium microsphere sample solution is firstly filled with the rubber hose, and then filled with the dielectrophoresis chip. At the same time, it is necessary to observe whether there are bubbles. The existence of bubbles will affect the movement direction and speed of the eutectic gallium indium microspheres. The advancing speed should not be too fast. If there are bubbles, the speed of the micro-injection pump should be reduced, and the solution should be introduced slowly to discharge the bubbles. If the bubbles cannot be discharged, the dielectrophoresis chip can only be removed and cleaned, then dried and operated again.

5) When it is observed that the eutectic gallium indium microsphere sample solution is completely filled with the dielectrophoresis chip and no bubbles are generated, adjust the flow rate of the micro-syringe pump, first observe and take pictures of the eutectic gallium indium microspheres under the condition of no power. motion track. After the observation and shooting are completed, connect the output end of the ultrasonic power amplifier to the copper wire on the dielectrophoresis chip, adjust parameters such as voltage and frequency, observe and record the movement of the eutectic gallium indium particle balls in the sorting area of the dielectrophoresis chip The statistical analysis of the particles was carried out with software such as ImageJ.

6) The eutectic gallium indium microparticles are captured by the microelectrode under the action of forward dielectrophoretic force, turn off the micro injection pump for transporting the sample solution, turn on the micro injection pump for transporting the medium solution, and disconnect the ultrasonic power amplifier from the medium at the same time. The connection of the electrophoresis chip makes the metal chromium electrode lose its adsorption and capture effect, and the medium solution enters the sorting area from the bottom inlet to flush out the eutectic gallium indium particles captured by the metal chromium electrode, so as to realize the collection of the eutectic gallium indium particles selected by the separation .

By repeating steps 3) to 6) with different medium solutions, the influence of the conductivity of different medium solutions on the continuous separation efficiency of eutectic gallium indium particles can be studied. It should be noted that when the ultrasonic power amplifier is turned off, the connection between the ultrasonic power amplifier and the dielectrophoresis chip should be disconnected first, so as to avoid the generation of many bubbles and damage to the electrode due to the sudden voltage change of the ultrasonic power amplifier.

After all operations are completed, clean the dielectrophoresis chip with deionized water, observe the chip under a microscope after cleaning, dry the chip and package it for next use. Finally, close all instruments and clean up the operating platform.

For the single eutectic eutectic gallium indium particle sorting results are:

The parameters of frequency 100kHZ, suspension flow rate 50μl/min, voltage 20V, and suspension conductivity 7μS/m are the optimal parameters. At this time, the separation efficiency of eutectic gallium indium particles is as high as 99%. The physical map of sorting is shown in Figure 14.

2. Sorting and capture of EGaln-red blood cell mixed solution;

The sample solution in this test process is a mixed sample solution, and the configuration method of the mixed sample solution is as follows: the particles used are mainly eutectic gallium indium with a radius of 15um and red blood cells with a radius of 5um.

Preparation of eutectic gallium indium suspension solution: Take 100ml of deionized water and add it to a beaker, add 10ul eutectic gallium indium solution with a pipette gun, dilute 100 times, shake for 1min to make the diluted solution eutectic gallium indium evenly distributed, and get Desired eutectic Gallium Indium pellet suspension solution.

Preparation of red blood cell suspension solution: first prepare 100ml of normal saline, then take a drop of blood from the human body with a blood collection needle, add it to the prepared normal saline to dilute, shake for 1 minute to make the red blood cells in the diluted solution evenly distributed, and obtain the required red blood cell suspension solution.

Preparation of mixed sample solution: use a pipette to extract 10 μl of eutectic gallium indium microparticle pellets, put them in a 5ml centrifuge tube and add PBS phosphate buffered saline solution to 5ml, then use a pipette to extract 50μl of red blood cells, and place them in another 5ml Add PBS phosphate-buffered saline to the centrifuge tube to 5ml, mix and prepare repeatedly in different proportions, and finally prepare a mixed sample solution with a ratio of eutectic gallium indium microspheres to red blood cells at a ratio of 1:100, as shown in Figure 15 .

The dielectrophoresis chip sorts the eutectic gallium indium microspheres and red blood cells and operates as follows:

1) Take out the packaged and bonded DEP chip, check whether the DEP chip is damaged, and use an electric soldering iron to solder the two copper wires to the two pins of the DEP chip respectively, and then use a multimeter to check whether the soldering is correct. Qualified, the dielectrophoresis chip can be used only after the test is qualified.

2) Turn on the power of the operating system and check whether the instruments in the operating system can work normally. After the inspection is correct, fix the dielectrophoresis chip under the microscope and adjust the microscope so that the sorting area of the dielectrophoresis chip can be clearly seen on the computer monitor.

3) Connect the 5ml syringe to the infusion tube, then shake the mixed sample solution to make it evenly mixed, quickly draw the syringe in reverse, the mixed sample solution is in the infusion tube, hang the infusion tube vertically, and keep the infusion tube away from One end of the syringe is connected to the inlet of the microinjection pump, and the outlet of the microinjection pump is communicated with the upper inlet of the dielectrophoresis chip through a rubber hose, that is, the upper inlet is connected to the mixed sample solution. Take another 5ml syringe, draw 5ml of PBS phosphate buffered saline as the medium solution, connect the syringe to the inlet of another micro-injection pump through the infusion tube, and the outlet of the micro-injection pump is connected to the dielectrophoresis chip through a rubber hose The lower inlet is connected to PBS phosphate-buffered saline.

4) After the particle delivery device is connected, first turn on the micro-injection pump that transports the mixed sample solution, and then slowly push the syringe so that the mixed sample solution is first filled with the rubber hose to discharge air bubbles, and the flow rate of the mixed sample solution is set to 50 μl/min, so that It slowly fills the dielectrophoresis chip. At the same time, pay attention to whether there are bubbles. The existence of bubbles will affect the direction and speed of the eutectic gallium indium microspheres. If there are bubbles, use tweezers to gently press the bubbles The position of the corresponding dielectrophoresis chip above does not affect the operation until the bubbles are discharged or become very small.

5) When it is observed that the eutectic gallium indium particle balls are completely filled with the chip and no air bubbles are generated, first observe and photograph the movement track of the eutectic gallium indium particle balls without power on. After the observation and shooting are completed, connect the output terminal of the ultrasonic power amplifier to the copper wire on the dielectrophoresis chip, adjust the peak-to-peak value of the function signal generator to 5V, the frequency to 100kHz, the waveform to sine wave, the phase to 0, and the output channel switch to Turn on, send the signal to the ultrasonic power amplifier, and then adjust the ultrasonic power amplifier to make the output voltage 15V, observe and record the movement of eutectic gallium indium particle pellets and red blood cells in the sorting area of the dielectrophoresis chip, using ImageJ, etc. The software performs statistical analysis of the particles.

6) Use microfluidic dielectrophoresis chip to sort red blood cells and eutectic gallium indium particle balls. The dielectrophoretic force of the dielectrophoresis chip is much smaller than the hydrodynamic force of the solution in the dielectrophoresis chip. Under the action of the hydrodynamic force, the red blood cells flow out from the outlet with the solution; turn off the micro-injection pump for transporting the mixed sample solution, and turn on the micro-injection pump for transporting the medium solution. Disconnect the connection between the ultrasonic power amplifier and the dielectrophoresis chip at the same time, so that the metal chromium electrode loses the adsorption and capture effect. The flow rate of the medium solution is 50 μl/min. The medium solution is PBS phosphate buffered saline solution with a conductivity of 15 μS/min. Enter the sorting area to punch out the eutectic gallium indium particles captured by the metal chromium electrode, realize the collection of the sorted eutectic gallium indium particles, and then achieve the purpose of sorting.

It should be noted that first disconnect the ultrasonic power amplifier from the dielectrophoresis chip, and then turn off the ultrasonic power amplifier. Finally, clean the dielectrophoresis chip with deionized water, observe the chip under a microscope after cleaning, and dry the chip. Package it for next use. Then turn off all instruments and clean the platform.

<Force Analysis of Particles in Dielectrophoresis Chip>

In terms of EGaln sorting and capture: the force analysis of particles in the microfluidic dielectrophoresis chip is as follows:

The force on the particle in the DEP chip includes not only the DEP force, but also forces such as buoyancy, gravity, hydrodynamic force, fluid resistance, and intermolecular forces. Compared with dielectrophoretic force and hydrodynamic force, other forces are too small, so the dielectrophoretic force F _DEP and hydrodynamic force F _l suffered by particles are mainly considered. The schematic diagram of particle force is shown in Figure 9, so that The resultant force F experienced by the particles in the dielectrophoretic chip can be expressed as:

When no AC electric field and flow velocity are applied (i.e. E=0, V=0), the particles will not move in a state of equilibrium in the DEP chip; Under the action of electrophoretic force F _DEP and hydrodynamic force F _l will move, then the equation of motion of particles in the dielectrophoretic chip can be expressed by the following formula:

为粒子在介电泳芯片中所受的合力，单位为N。In the above formula: m _m is the mass of the particle, the unit is kg; v _m is the moving speed of the particle in the dielectrophoresis chip, the unit is m/s;

is the resultant force on the particle in the dielectrophoresis chip, in N.

Since the particles used are spherical particles, their mass is:

In the above formula, r is the radius of the particle, the unit is m; ρ _m is the density of the particle, the unit is kg/m ³ .

In the microfluidic dielectrophoresis chip, the particle displacement x _m (t) can be expressed as:

Ignoring the acceleration process of particles in the microfluidic field, only considering the uniform motion of particles, then in the x direction:

F _DEP sinθ＝F _l

F _l ＝6πμηr(v _mx -v _m )

In the z direction there are:

F _DEP cosθ＝F _drag

F _drag ＝6πηrv _mz

From this, it can be obtained that the moving speed of particles in the microfluidic dielectrophoresis chip is:

v _mx represents the moving speed of the particle in the x direction in the dielectrophoretic chip, v _mz represents the moving speed of the particle in the z direction in the dielectrophoretic chip, η represents the pseudo-speed of the particle, θ represents the resultant force F and the hydrodynamic force F _l angle.

<Sorting results of EGaln-red blood cell mixed solution>

The present invention adopts a wave-shaped microelectrode, adjusts the flow rate of the micro-injection pump to 150um/s, adjusts the function signal generator to 0.04V and 500kHz, and then mixes eutectic gallium indium and red blood cells in a 5ml syringe The sample solution is connected to the upper inlet. After all equipment debugging is completed, turn on the micro-injection pump for transporting the sample solution, turn off the micro-injection pump for transporting the medium solution, and observe the movement state of the particles in the microscope on the computer, as shown in Figure 10 .

It can be seen from Figure 10 that the eutectic GaIn and red blood cells around the electrodes were scattered before power-on, but it can be clearly seen from Figure 11 that the eutectic GaIn around the electrodes was captured by the metal chromium electrode after power-on, while the blood The red blood cells pass directly through the metal chromium electrode, and in Figure 12 there are a large number of red blood cells around the outlet of the channel. Afterwards, we used ImageJ software to count and count the three pictures in Figure 10, Figure 11, and Figure 12, and obtained the ratio of eutectic gallium indium and red blood cells in the solution as shown in Figure 13:

It can be seen from Figure 13 that there is only 10% eutectic gallium indium in the solution before power is applied, but after power is applied, eutectic gallium indium is captured by the electrode, and red blood cells can still pass through, so the proportion of eutectic gallium indium in the solution is increase, and the ratio of red blood cells in the solution at the outlet is as high as 100%, achieving the effect of separating the eutectic gallium indium and red blood cells.

Claims (10)

1. The micro-fluidic dielectrophoresis chip is characterized by comprising a microelectrode and a microchannel, wherein the microelectrode comprises a glass substrate and two chromium metal electrodes positioned on the glass substrate, each chromium metal electrode comprises a wavy section and a pin section connected to the end part of the wavy section, the wavy sections of the two chromium metal electrodes are parallel to each other, and the pin sections of the two chromium metal electrodes deviate from each other; the microchannel is a PMDS cover plate with an inwards concave channel on the lower surface, the microchannel is bonded and packaged with the microelectrode through PMDS, and the wave sections of the two metal chromium electrodes are positioned in the inwards concave channel of the microchannel.

2. A microfluidic dielectrophoresis chip according to claim 1, wherein the width of the wavy section is 100 μm, the spacing between the wavy sections of the two chromium metal electrodes is 100 μm, and the width of the sorting region formed by the wavy sections is 850 μm.

3. A microfluidic dielectrophoresis chip according to claim 1, wherein the microchannel comprises a channel body, two inlets at one end of the channel body and one outlet at the other end of the channel body, the channel body comprising an inlet section, a sorting section and an outlet section which are in communication in sequence, one end of the sorting section being in communication with the two inlets through the two inlet sections respectively, and the other end of the sorting section being in communication with the outlet through the one outlet section.

4. A microfluidic dielectrophoresis chip according to claim 3, wherein the depth of the recessed channel is 0.5mm, the width of the sorting section is 2mm, and the width of both the inlet section and the outlet section is 1mm.

5. A method of fabricating a microfluidic dielectrophoresis chip according to claim 3, comprising the steps of

S1: manufacturing a microelectrode: manufacturing a chromium metal electrode on a glass substrate by photoetching and wet etching to form a microelectrode;

s2: manufacturing a micro-channel: taking another glass substrate, enclosing a containing cavity on the glass substrate, fixing the microchannel male die in the containing cavity, pouring PDMS into the containing cavity and carrying out curing treatment to form a PMDS cover plate; taking the PMDS cover plate out of the accommodating cavity, and taking out the male die to form a micro-channel, wherein the male die is positioned in the concave channel;

s3: bonding and packaging a microelectrode and a microchannel: and (3) coating PMDS on the lower surface of the PMDS cover plate, covering the PMDS on a glass substrate on which the chromium metal electrode is positioned, so that the wavy section of the chromium metal electrode is positioned in the concave channel, and curing to obtain the microfluidic dielectrophoresis chip.

6. A method according to claim 5, wherein in step S2, the receiving chamber is defined by a glass substrate and four glass slides, and the four glass slides are connected end to end and are bonded to the glass substrate.

7. A method according to claim 5, wherein in step S2, the male mold comprises a plate blank and projections fixed to both ends of the plate blank for forming the inlet and outlet of the microchannel, respectively.

8. A method according to claim 7, wherein in step S2, the height of the PDMS in the cavity is not higher than the height of the protrusions.

9. Use of a microfluidic dielectrophoresis chip according to claim 3 in the separation of liquid metal particles.

10. The application of the microfluidic dielectrophoresis chip in liquid metal particle sorting according to claim 9, wherein firstly, a solution to be sorted containing liquid metal particles is introduced into the microchannel from one inlet of the microchannel, after the recessed channel is filled with the solution to be sorted, an ultrasonic power amplifier connected with the microfluidic dielectrophoresis chip and a function signal generator connected with the ultrasonic power amplifier are started, parameters of the function signal generator are adjusted, an inhomogeneous electric field is provided to the microfluidic dielectrophoresis chip after the parameters are amplified by the ultrasonic power amplifier, the liquid metal particles are captured by the metal chromium electrode under the action of the inhomogeneous electric field, then the inlet into which the solution to be sorted is closed, a medium solution is introduced from the other inlet of the microchannel, the ultrasonic power amplifier is disconnected, and the liquid metal particles flow out of the recessed channel along with the medium solution after the metal chromium electrode is captured, so as to realize sorting.

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