CN118956534A - Application of pine pollen micro-robot combined micro-fluidic chip in cell capture - Google Patents

Abstract

The invention belongs to the technical field of microfluidics, and particularly relates to application of a pine pollen micro-robot combined microfluidic chip in cell capture, wherein the pine pollen micro-robot is used for sputtering gold pine pollen, the microfluidic chip is filled with a pine pollen micro-robot and a cell solution with conductivity, and when alternating current signals are applied to the microfluidic chip, fluid around the pine pollen micro-robot generates induced charge electroosmotic vortex so that cells in the cell solution move to cavities of the pine pollen micro-robot, and further cell capture is realized. The invention can realize the high-efficiency capturing of other micron-sized cell structures such as blood cells and the like by inducing the charge electroosmosis (ICEO) technology, solves the problems that the traditional capturing technology relies on modes such as antibody marking, mechanical capturing and the like, reduces the complexity of operation and the capturing difficulty, and improves the capturing success rate and the raw material utilization rate.

Description

Application of pine pollen micro-robot combined micro-fluidic chip in cell capture

Technical Field

The invention belongs to the technical field of microfluidics, and particularly relates to application of a pine pollen micro-robot combined microfluidic chip in cell capture.

Background

Cell analysis is a key element in biomedical research, and reveals disease mechanisms and potential therapeutic methods by delving into the vital basic unit of cells. The study of the survival mechanism and basic structure of cells, which are the basic units of life, is crucial to understanding the therapeutic methods and causes of diseases, which helps to improve human life and quality of life. With the development of technology, cell analysis has evolved from traditional microscopic observations to modern high-throughput cell sequencing, which is capable of rapidly analyzing gene expression of a large number of cells, providing more abundant data for disease diagnosis and treatment. The advent of microfluidic technology has also created new possibilities for cell analysis. Compared with the traditional flow cytometry and other analysis tools, the microfluidic technology stands out by the characteristics of simple structure, low cost and high customization.

Cell extraction technology is the basis of cell analysis operations, and currently there are two main methods: traditional micromanipulation techniques and microfluidic technology based cell extraction. Micromanipulation techniques manually select and extract cells under a microscope using micropipettes, while intuitive and accurate to operate, present a risk of contamination that can be time consuming, requiring extensive sample preparation and open experimental environments. Based on the application of the microfluidic technology, the method is more common in cell capturing and micropore (micro-well) methods with micro-valve control. For micro-valve controlled cell capture, the method uses micro-valves on a microfluidic chip to control the flow path and velocity of the fluid, thereby achieving accurate positioning and capture of cells. The design of the queue valve is often difficult to adapt to a plurality of different types of cells, and the micro-valve control itself requires an accurate control algorithm and high manufacturing accuracy, the overall device is high in false cost, and frequent operation of the micro-valve may damage the cells or affect the activity of the cells. The principle of cell extraction by using a micropore (micro-well) method is simpler, single cells are captured and extracted by designing a tiny hole or trap structure, the cells enter the independent micropores due to gravity, and the cells outside the micropores are washed away along with a medium. Although this method can capture enough cells at a time for analysis, the cells may only occupy a portion of the microwells, and thus the number of cells that a single microwell may capture is not certain, which can present difficulties for subsequent molecular analysis of the cells.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides the application of the pine pollen micro-robot combined micro-fluidic chip in cell capturing, single target cells are captured in a target cell-containing medium by utilizing the micro-robot, and the capturing and release of the single cells can be controlled by adjusting external voltage and frequency signals, so that the risk of biological pollution of directly contacting the target cells is greatly reduced, and a more reliable and efficient technical platform is provided for molecular structure analysis and biomedical research of the cells.

The technical scheme provided by the invention is as follows:

The invention provides an application of a pine pollen micro-robot combined micro-fluidic chip in cell capturing, wherein the pine pollen micro-robot is used for sputtering pine pollen of gold, the micro-fluidic chip is filled with the pine pollen micro-robot and a cell solution with conductivity, and when alternating current signals are applied to the micro-fluidic chip, fluid around the pine pollen micro-robot generates induced charge electroosmotic vortex so that cells in the cell solution move to cavities of the pine pollen micro-robot, and further cell capturing is realized.

Furthermore, the cells in the cell solution move to the cavity of the pine pollen micro-robot and are subjected to the dielectrophoresis force.

Further, the pine pollen micro-robot is provided with two cavities with nearly spherical air sac structures, and the nearly spherical air sac structures of the two cavities generate locally enhanced electric field intensity, so that cells are accelerated to move to the cavities.

Further, the voltage of the applied alternating current signal is 10-15V, and the frequency is 100-500 HZ.

Further, the cell suspension with the conductivity comprises a solution A and a solution B, wherein the solution A is absolute ethyl alcohol and tween solution according to the volume ratio of 9: the active agent solution prepared in the step (1) is a cell buffer solution with the conductivity of 1-6 mS/m prepared by dissolving potassium chloride in deionized water, and the volume ratio of the solution A to the solution B is 1:99.

Further, the microfluidic chip comprises a conductive cover plate and a base plate, a fluid inlet and a fluid outlet are arranged above the cover plate, an intermediate cover layer for accommodating the pollen pini micro-computer and the cell suspension is arranged between the cover plate and the base plate, electrodes are respectively arranged on the cover plate and the base plate, and the electrodes are connected with a signal generator for applying alternating current signals through wires.

Furthermore, the cover plate and the bottom plate are both made of glass and are overlapped in parallel and staggered, and the indium tin oxide coating is coated on one surface of each cover plate and each bottom plate, which is in contact with the middle covering layer.

Further, the electrode is a metal copper film which is adhered along the edge of the wide edge of the non-overlapped part of the cover plate and the bottom plate and is arranged towards one side of the middle covering layer.

Further, the length of the non-overlapped part of the cover plate and the bottom plate is larger than the width of the metal copper film stuck on the two sides, and the length of the metal copper film is smaller than the width of the cover plate and the bottom plate.

Further, the middle covering layer is a polyethylene terephthalate film provided with four surrounding grooves, and the length and the width of the polyethylene terephthalate film are smaller than those of the cover plate and the bottom plate.

Advantageous effects

The invention designs a micro-robot capable of generating induced potential by utilizing metal sputtered pollen pini, single specific target cells are directly captured in a cell medium solution, the whole micro-fluidic chip structure is sandwich-type, the prepared sputtered pollen pini and yeast cell solution with conductivity are injected into the micro-fluidic chip from a fluid inlet of ITO glass at the top in the experiment, and free electrons in the solution are induced to migrate to form dipole moment and conductive current through alternating current signals loaded on the top layer and the bottom layer, so that the phenomenon of double electric layers appears on the surface of the micro-robot. After reaching steady state, because the electric double layers with opposite polarities exist on the surface of the sputtered pollen pini, the positive and negative electric layers can move along different directions of the electric field lines, and then a convection effect, namely ICEO (induced charge electroosmosis) vortex is formed in the channel space, so that the fluid near the surface of the pollen pini micro-robot generates electroosmosis vortex, and the nearby target cells are pulled to the robot cavity. Meanwhile, due to the fact that dielectric coefficients are different due to the fact that polarization properties of target cells and cell medium solutions are different, the target cells are subjected to the effect of dielectrophoresis, and the special geometric structure of the pine pollen micro-robot enables the cavity to easily obtain extremely large electric field intensity, and the approaching speed of the target cells to the needle point is accelerated.

The invention can realize the high-efficiency capturing of other micron-sized cell structures such as blood cells and the like by inducing the charge electroosmosis (ICEO) technology, solves the problems that the traditional capturing technology relies on modes such as antibody marking, mechanical capturing and the like, reduces the complexity of operation and the capturing difficulty, and improves the capturing success rate and the raw material utilization rate.

Drawings

Fig. 1 is an overall structural view of a microfluidic chip of the present invention.

Fig. 2 is a schematic diagram of components of a microfluidic chip of the present invention.

Fig. 3 is a top view of a microfluidic chip of the present invention.

Fig. 4 is a front view of a microfluidic chip of the present invention.

Fig. 5 is a partial enlarged view of the microfluidic chip of the present invention.

FIG. 6 is a schematic diagram of a pollen Pini micro-robot according to the present invention.

FIG. 7 is a diagram of a pine pollen micro-robot according to the present invention.

FIG. 8 is a graph of the electric field distribution around a pollen pini micro-robot at low frequencies.

FIG. 9 is a graph of the electric field distribution around a pollen pini micro-robot at high frequency.

Figure 10 is a fluid swirl chart around a pollen pini micro-robot at low frequency.

Reference numerals illustrate: 1. a cover plate; 2. a bottom plate; 3. an intermediate cover layer; 4. an electrode; 5. a fluid inlet; 6. a fluid outlet; 7. sputtering pollen Pini; 8. yeast cells.

Detailed Description

Example 1

As shown in fig. 1-5, an embodiment of the present invention provides a microfluidic chip, where the microfluidic chip includes a conductive cover plate 1 and a base plate 2, a fluid inlet 5 and a fluid outlet 6 are disposed above the cover plate, an intermediate cover layer 3 for accommodating a pollen pini micro-robot and a cell suspension is disposed between the cover plate 1 and the base plate 2, electrodes 4 are disposed on the cover plate 1 and the base plate 2, respectively, and the electrodes 4 are connected to a signal generator applying an ac electrical signal through wires.

In this embodiment, the cover plate 1 and the bottom plate 2 are both made of glass and are overlapped in parallel and staggered, and the surface of each of the cover plate 1 and the bottom plate 2, which is in contact with the middle cover layer 3, is coated with an indium tin oxide coating.

In this embodiment, the electrode 3 is a metal copper film, and the metal copper film is adhered along the edge of the wide edge of the non-overlapped portion of the cover plate 1 and the bottom plate 2 and is disposed toward the side of the middle cover layer 3.

In this embodiment, the length of the non-overlapped portion of the cover plate 1 and the bottom plate 2 is greater than the width of the metal copper thin film stuck on both sides, and the length of the metal copper thin film is smaller than the length and the width of the cover plate 1 and the bottom plate 2.

In this embodiment, the middle cover layer 3 is a polyethylene terephthalate film with four grooves, the length and width of the polyethylene terephthalate film are smaller than those of the cover plate and the base plate, and the PET film has good ductility and good adhesion with glass, can be directly contacted with biological samples, and does not cause adverse reactions.

Specifically, the glass has a length of 30 mm, a width of 30 mm and a height of 1mm, and indium tin oxide is coated on the whole outer surface of one side of the glass, and the thickness is 0.5 mu m; the middle coating layer 3 is a PET film, the length is smaller than that of glass, the length is 18mm, the width is 28mm, the thickness is 0.2mm, part of the middle layer is removed, and four surrounding grooves with the length of 15mm and the width of 24 mm are formed; the metal copper film is 10mm long and 4mm wide, is tightly attached to the edge of one side of the conductive glass with the Indium Tin Oxide (ITO) coating, and is led out by a connecting wire; the radius of the inlet and outlet channels is 1mm.

The preparation method of the microfluidic chip comprises the following steps:

The ITO glass is cleaned and wiped by deionized water, the PET film is cut to the specified size, the protective film on one side of the film is torn off, the glass is clung to the center of the ITO side of the glass, the glass is ensured to be placed in a plasma bonding machine in an upward direction, the power of the plasma bonding machine is set to be 10W, the oxygen switch is set to be 8-10 NI/h, and the treatment is carried out to 32 s. Cutting part of PET by using a nicking tool, forming a four-peripheral groove in the center of the film, tearing off the protective film of the other half of PET, aligning one side of the other piece of glass with ITO downwards and staggering a certain position to be clung to the PET film (ensuring that the PET film is covered by two layers of glass, staggering the glass by a certain position, namely, extending the edges of the glass to be not opposite), performing plasma bonding again, standing for ten minutes after taking out, and observing whether bubbles exist at the edges of the four-peripheral groove (the bubbles represent unbonded work); and finally transferring the micro-fluidic chip to a heating carbon plate, pressing the micro-fluidic chip on the surface by using a heavy object, and setting the temperature to 80 ℃ for two hours to obtain the micro-fluidic chip bonded.

Example 2

1. Preparation of sputtered pollen Pini

1) Mixing 1g pollen Pini with 20ml absolute ethanol, placing in an ultrasonic instrument for 5-10min, filtering the mixture with filter paper, repeating the above operation for 3 times, and collecting a small amount of sample with a pipette under microscope for removing impurities and dispersing pollen Pini.

2) Mixing the obtained pollen Pini with 20ml absolute ethanol again, placing in an ultrasonic instrument for 5min, immediately extracting 20 μl of the mixed solution with a pipette, dripping onto glass slide for several times, and spreading the mixed solution on glass slide uniformly by plate scribing method.

3) The glass slide is taken out after being placed in a drying box for heat preservation for 20-30 minutes at the temperature of 60 ℃, and is naturally cooled to the room temperature, so that the pollen pini scattering condition can be observed by naked eyes, the stacking of a plurality of layers is avoided, and if necessary, the stacking blocks are removed by using an adhesive tape.

4) Placing the pollen Pini slide glass in an ion sputtering instrument, sputtering metal gold, screwing a valve, pumping air in the sputtering chamber to a low vacuum environment (10 mmHg), starting sputtering for about 1-2min, adjusting the direction of the slide glass after the sputtering is completed, and re-sputtering once.

5) Putting a culture dish into a glass slide subjected to sputtering, adding a proper amount of absolute ethyl alcohol until the glass slide is completely immersed, slightly scraping pollen Pini (which can be observed under a microscope, and a layer of dark green layer is covered by transparent cytoplasm of the original pollen Pini) by using a hairbrush, slowly introducing the mixed solution into a filter paper funnel, completely leaving solid at the bottom of the funnel, allowing absolute ethyl alcohol to drop completely, slowly dripping deionized water at the edge of the filter paper until the solid is immersed, and repeatedly dripping for 2-3 times to complete cleaning.

2. Sample preparation:

to prevent cells from sticking to the glass substrate or edge PET during the experiment, thereby affecting experimental observations, an active agent solution may be formulated to inhibit cell attachment.

The preparation method of the active agent solution comprises the following steps: preparing absolute ethyl alcohol and tween solution in a volume ratio of 9:1 to obtain an active agent A solution, and preventing cell adhesion in the experimental process; KCL is dissolved in deionized water, and a conductivity meter is used for detection when stirring, and the solution is adjusted to be suitable conductivity (1-6 mS/m) to serve as a cell buffer solution B, so that the osmotic pressure inside and outside cells is balanced; the a solution and the B solution were then mixed at 1:99 was mixed in the volume ratio to prepare a C solution as a cell culture solution for experiments.

20 Mg dry yeast powder was mixed with 5ml deionized water and placed in an sonicator for 5min and heated in an oven at 50℃for two hours. Then, 1ml of the yeast suspension was placed in a centrifuge tube with a pipette, centrifuged and repeated three times with the B solution to remove metabolic waste and impurities in the yeast solution. And finally, the cleaned saccharomycetes are placed in a C solution with corresponding conductivity again. The prepared solutions were concentration-formulated with a cell counter plate.

3. Experimental operation:

1) The prepared sputtered pollen pini 7 and yeast cell solution with conductivity are injected into a microfluidic chip from a fluid inlet 5 of ITO glass at the top, a microscope, a computer and a CCD camera are sequentially opened, a metal copper film is tightly attached to the extending edges (the upper glass layer and the lower glass layer are respectively attached to one side of the ITO-containing side of the microfluidic chip) of the two glass layers, the PET film cannot be touched, connecting wires are led out, the sputtered pollen pini and the yeast cell solution with conductivity are placed on an objective table of a fluorescent microscope, the objective is reasonably adjusted according to experimental requirements, and the chip position and focal length are fixed.

2) Connecting an output port of a signal generator to a lead led out from a chip, turning on a power supply of the signal generator, and adjusting the voltage and frequency (10-15V; 100-500 HZ), the top end of the cavity of the sputtered pollen pini is induced to produce electroosmosis effect of induced charges to adsorb yeast cells 8, thus completing the capture of the cells.

Figures 6-7 show the unique structure of pollen pini, the hollow of which makes most of the balloon present a balloon-up, cytoplasmic-down posture when naturally floating in water, and when sputtering occurs, metallic substances will preferentially deposit on the top of the balloon and partially exposed cytoplasm, these areas thus having a much higher electrical conductivity than the surrounding environment.

Under the action of an alternating current electric field, polarized dielectric particles (pine pollen micro-robot) can generate induced charges under the action of dipole moment, voltage can be concentrated in places with larger conductivity, namely, sputtering parts of the pine pollen have a large amount of induced charges (similar to electrodes), at the moment, a vertical electric field can deviate around the sputtering parts, the direction is that the top of an air bag points to middle cytoplasm, under the action of the non-uniform alternating current electric field, a current path can also twist to form a local high current density area, a large amount of non-uniform temperature rise is generated in a solution in the areas due to the joule effect of the current, so that a temperature gradient is formed, the conductivity gradient and the dielectric coefficient in the solution are also formed due to the existence of the fluid temperature gradient, and fluid flows along the gradient direction under the action of the alternating current electric field, namely, an alternating current electric heating coupling effect is generated; meanwhile, the target cells are polarized to generate induced charges under the action of an electric field, movement, namely Dielectrophoresis (DEP) is generated under the action of a non-uniform alternating current electric field, the movement of the target cells is controlled by two forces, the dominant forces are different under different conditions, for example, larger yeast cells are more subjected to dielectrophoresis force, smaller yeast cells are more subjected to fluid vortex, and the fluid vortex is generated only when the voltage is concentrated under low frequency.

As shown in fig. 8, red lines are electric field lines, the color of the region represents the distribution of electric charges, at low frequency, the electric charges are concentrated on two sides of the top of the air bag, namely, red and green regions in the figure, which are the necessary conditions for fluid vortex generation, and the electric field lines inside the air bag are vertical, the surrounding electric field is offset, so that a non-uniform electric field is generated, especially in the middle and two sides of pollen cells, the directions of the fluid vortex can be seen in fig. 10, the directions of the fluid vortex directions are respectively pointed to the middle and two sides of the cells from the top of the air bag, and the cells move along the direction of the fluid vortex (blue line) in fig. 10 and are finally captured near the cells. As shown in fig. 9, at high frequency, the electric charges are almost uniformly distributed, the electric field bypasses the cells and has the same direction, and the fluid vortex is small or even disappears, and the trapping of the cells is weakened only by inducing the attraction of the electric charges.

The invention designs a microcomputer robot capable of generating induced potential by utilizing metal sputtered pollen pini, and single specific target cells are directly captured in a cell medium solution. The whole micro-fluidic chip structure is sandwich, the upper and lower layers are covered by conductive ITO glass, the middle layer is a working area framed by PET film, and the ITO glass on the top layer is provided with an inflow and outflow port containing cell media. When the ITO glass of the upper layer and the lower layer is added with alternating current signals, induced charge electroosmotic flow (ICEO) is formed on the surface of the micro-robot in cell medium solution, micro-eddies in the fluid can guide cells near two cavities of pollen pini to move to the cavities, and in addition, due to the effect of electric field gradient, target cells are subjected to the effect of Dielectrophoresis (DEP). Due to the unique geometric shape of pollen Pini, i.e. the nearly spherical balloon structure, very high local electric field strength will be generated at the two cavities, which will accelerate attracting cells towards the cavities. Compared with the traditional cell capturing scheme, the scheme has low manufacturing cost and simple structure, and avoids the complicated microchannel design process; and the control process is completed by electric control, so that the probability of the target cells receiving external pollution is reduced to a great extent.

Claims (10)

1. The application of the pine pollen micro-robot combined micro-fluidic chip in cell capturing is characterized in that the pine pollen micro-robot is pine pollen sputtered with gold, the micro-fluidic chip is filled with the pine pollen micro-robot and cell solution with conductivity, and when alternating current signals are applied to the micro-fluidic chip, fluid around the pine pollen micro-robot generates induced charge electroosmotic vortexes so that cells in the cell solution move to cavities of the pine pollen micro-robot, and further cell capturing is achieved.

2. The use of a pine pollen micro-robot combined micro-fluidic chip in cell capture according to claim 1, wherein cells in the cell solution are also subjected to dielectrophoresis forces moving towards the pine pollen micro-robot cavity.

3. The use of a pine pollen micro-robot in combination with a cell capture of a microfluidic chip according to claim 1, wherein the pine pollen micro-robot has two cavities of nearly spherical air-bag structure which produce locally enhanced electric field strength to accelerate the movement of cells to the cavities.

4. The application of the pine pollen micro-robot combined micro-fluidic chip in cell capturing according to claim 1, wherein the voltage of the applied alternating current signal is 10-15V, and the frequency is 100-500 HZ.

5. The application of the pine pollen micro-robot combined micro-fluidic chip in cell capturing according to claim 1, wherein the cell solution with the conductivity comprises solution A and solution B, wherein the solution A is absolute ethyl alcohol and tween solution in a volume ratio of 9: the active agent solution prepared in the step (1) is a cell buffer solution with the conductivity of 1-6 mS/m prepared by dissolving potassium chloride in deionized water, and the volume ratio of the solution A to the solution B is 1:99.

6. The use of a pine pollen micro-robot combined micro-fluidic chip in cell capturing according to claim 1, wherein the micro-fluidic chip comprises a conductive cover plate and a bottom plate, a fluid inlet and a fluid outlet are arranged above the cover plate, an intermediate cover layer for containing a pine pollen micro-robot and a cell suspension is arranged between the cover plate and the bottom plate, and electrodes are respectively arranged on the cover plate and the bottom plate and connected with a signal generator for applying alternating current signals through wires.

7. The application of the pine pollen micro-robot combined micro-fluidic chip in cell capturing according to claim 6, wherein the cover plate and the bottom plate are both made of glass and are overlapped in parallel and staggered mode, and the surface, which is contacted with the middle covering layer, of each cover plate and each bottom plate is coated with an indium tin oxide coating.

8. The use of pine pollen micro-robot combined micro-fluidic chip as claimed in claim 7, wherein the electrode is a metallic copper film adhered along the edge of the broadside of the non-overlapping portion of the cover plate and the base plate and disposed toward the side of the middle cover layer.

9. The application of the pine pollen micro-robot combined micro-fluidic chip in cell capturing according to claim 8, wherein the length of the non-overlapped part of the cover plate and the bottom plate is larger than the width of the metal copper film stuck on two sides, and the length of the metal copper film is smaller than the width of the cover plate and the bottom plate.

10. The application of the pine pollen micro-robot combined micro-fluidic chip in cell capturing according to claim 6, wherein the middle cover layer is a polyethylene terephthalate film with four surrounding grooves, and the length and the width of the polyethylene terephthalate film are smaller than those of the cover plate and the bottom plate.