Application of a pine pollen microrobot combined with a microfluidic chip in cell capture

Abstract

This invention belongs to the field of microfluidics, and particularly relates to the use of a pine pollen microrobot combined with a microfluidic chip for cell capture. The pine pollen microrobot is gold-sputtered pine pollen, and the microfluidic chip is injected with the pine pollen microrobot and a conductive cell solution. When an alternating current signal is applied to the microfluidic chip, the fluid surrounding the pine pollen microrobot generates an induced charge electroosmotic vortex, causing cells in the cell solution to move toward the cavity of the pine pollen microrobot, thereby achieving cell capture. Using induced charge electroosmosis (ICEO) technology, this invention enables the efficient capture of blood cells and other micron-sized cell structures. This solves the problem of traditional capture techniques relying on antibody labeling and mechanical capture, reduces operational complexity and capture difficulty, and improves capture success rate and raw material utilization.

Description

Technical Field

[0001] The present invention belongs to the field of microfluidics technology, and in particular relates to an application of a pine pollen microrobot combined with a microfluidics chip in cell capture. Background Art

[0002] Cell analysis is a key component of biomedical research, revealing disease mechanisms and potential treatments by deeply exploring cells, the fundamental building blocks of life. As the fundamental building blocks of life, studying the survival mechanisms and basic structure of cells is crucial for understanding the treatments and causes of disease, which in turn helps improve human lifespan and quality of life. With the development of technology, cell analysis has evolved from traditional microscopic observation to modern high-throughput cell sequencing, which can rapidly analyze the gene expression of large numbers of cells, providing richer data for disease diagnosis and treatment. The rise of microfluidics has also brought new possibilities to cell analysis. Compared with traditional analytical tools such as flow cytometry, microfluidics stands out for its simple structure, low cost, and high degree of customizability.

[0003] Cell extraction technology is fundamental to cell analysis. Currently, there are two main approaches: traditional micromanipulation and microfluidics-based cell extraction. Micromanipulation involves manual selection and extraction of cells using a micropipette under a microscope. While intuitive and precise, it is time-consuming, requires extensive sample preparation, and carries the risk of contamination from an open experimental environment. Common applications of microfluidics include microvalve-controlled cell capture and microwell (microtrap) methods. Microvalve-controlled cell capture uses microvalves on a microfluidic chip to control the flow path and velocity of fluid, enabling precise cell positioning and capture. Quake valve designs often struggle to adapt to diverse cell types. Furthermore, microvalve control requires precise control algorithms and high manufacturing precision, resulting in high device costs. Frequent manipulation of the microvalve can also damage cells or affect their viability. Microwell (microtrap) cell extraction, on the other hand, relies on a simpler principle: a tiny hole or trap structure is designed to capture and extract individual cells. Cells are drawn into individual microwells by gravity, while cells outside the microwells are washed away with the medium. Although this method can capture enough cells for analysis at one time, the cells may only occupy a part of the microwell, so the number of cells that may be captured by a single microwell is uncertain, which will bring difficulties to subsequent cell molecular analysis. Summary of the Invention

[0004] In response to the shortcomings of the existing technology, the present invention provides an application of a pine pollen microrobot combined with a microfluidic chip in cell capture. The microrobot is used to capture single target cells in a medium containing target cells, and the capture and release of single cells can be controlled by adjusting external voltage and frequency signals, greatly reducing the risk of biological contamination from direct contact with target cells, and providing a more reliable and efficient technical platform for molecular structure analysis of cells and biomedical research.

[0005] The technical solution provided by the present invention is as follows:

[0006] The present invention provides an application of a pine pollen microrobot combined with a microfluidic chip in cell capture. The pine pollen microrobot is pine pollen sputtered with gold. The microfluidic chip is injected with the pine pollen microrobot and a cell solution with electrical conductivity. When an alternating current signal is applied to the microfluidic chip, the fluid around the pine pollen microrobot generates an induced charge electroosmotic vortex, causing cells in the cell solution to move toward the cavity of the pine pollen microrobot, thereby achieving cell capture.

[0007] Furthermore, the cells in the cell solution move toward the cavity of the pine pollen microrobot and are also affected by dielectrophoretic force.

[0008] Furthermore, the pine pollen microrobot has two cavities with nearly spherical airbag structures. The nearly spherical airbag structures of the two cavities generate locally enhanced electric field strength, thereby accelerating the movement of cells toward the cavities.

[0009] Furthermore, the voltage of the applied AC signal is 10-15V, and the frequency is 100-500HZ.

[0010] Furthermore, the cell suspension with conductivity includes liquid A and liquid B, wherein the liquid A is an active agent solution prepared by dissolving anhydrous ethanol and Tween solution in a volume ratio of 9:1, and the liquid B is a cell buffer solution with a conductivity of 1~6mS / m prepared by dissolving potassium chloride in deionized water. The volume ratio of the liquid A to the liquid B is 1:99.

[0011] Furthermore, the microfluidic chip includes a conductive cover and a base, a fluid inlet and a fluid outlet are provided above the cover, an intermediate covering layer for accommodating pine pollen microrobots and cell suspensions is provided between the cover and the base, electrodes are respectively provided on the cover and the base, and the electrodes are connected to a signal generator that applies an alternating current signal through wires.

[0012] Furthermore, the cover plate and the bottom plate are both made of glass and are parallel and staggered and overlapped. The cover plate and the bottom plate are both coated with an indium tin oxide coating on the side in contact with the middle cover layer.

[0013] Furthermore, the electrode is a metal copper film, which is adhered along the edge of the wide side of the non-overlapping portion of the cover plate and the bottom plate and is arranged toward one side of the middle covering layer.

[0014] Furthermore, the lengths of the non-overlapping portions of the cover plate and the bottom plate are both greater than the widths of the metal copper films attached to both sides, and the lengths of the metal copper films are smaller than the widths of the cover plate and the bottom plate.

[0015] Furthermore, the middle covering layer is a polyethylene terephthalate film with four circumferential grooves, and the length and width of the polyethylene terephthalate film are both smaller than those of the cover plate and the bottom plate.

[0016] Beneficial effects

[0017] This study utilizes metal-sputtered pine pollen to design a microrobot capable of generating an induced electric potential. This microfluidic chip employs a sandwich-shaped structure, injecting a solution of pre-prepared sputtered pine pollen and conductive yeast cells into the microfluidic chip through the top ITO glass fluid inlet. Alternating current signals applied to the top and bottom layers induce the migration of free electrons in the solution, forming a dipole moment and a conduction current, resulting in the formation of a double electric layer on the microrobot's surface. Once steady state is reached, the presence of oppositely polarized double electric layers on the sputtered pine pollen surface causes the positive and negative layers to move in different directions along the electric field lines, generating convection within the channel space. This induced ICEO (induced charge electroosmotic flow) vortices, which in turn generate electroosmotic vortices in the fluid near the pine pollen microrobot's surface, drawing the target cells toward the robot's cavity. At the same time, due to the different dielectric constants caused by the difference in polarization properties between the target cells and the cell medium solution, the target cells will be affected by the dielectrophoretic force, and the special geometric structure of the pine pollen microrobot makes it easy to obtain a very large electric field strength in its cavity, which will accelerate the speed of the target cells approaching the needle tip.

[0018] The present invention can achieve efficient capture of blood cells and other micron-sized cell structures through induced charge electroosmosis (ICEO) technology, solving the problem of traditional capture technology relying on antibody labeling, mechanical capture, etc., reducing the complexity of operation and capture difficulty, and improving the capture success rate and raw material utilization. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is the overall structural diagram of the microfluidic chip of the present invention.

[0020] Figure 2 Schematic diagram of the components of the microfluidic chip of the present invention.

[0021] Figure 3 It is a top view of the microfluidic chip of the present invention.

[0022] Figure 4 It is a front view of the microfluidic chip of the present invention.

[0023] Figure 5 It is a partial enlarged view of the microfluidic chip of the present invention.

[0024] Figure 6 It is a schematic diagram of the pine pollen microrobot of the present invention.

[0025] Figure 7 This is a physical picture of the pine pollen microrobot of the present invention.

[0026] Figure 8 This is the electric field distribution around the pine pollen microrobot at low frequency.

[0027] Figure 9 This is the electric field distribution around the pine pollen microrobot at high frequency.

[0028] Figure 10 This is a diagram of the fluid vortex around the pine pollen microrobot at low frequency.

[0029] Explanation of the accompanying drawings: 1. Cover plate; 2. Bottom plate; 3. Intermediate covering layer; 4. Electrode; 5. Fluid inlet; 6. Fluid outlet; 7. Sputtering pine pollen; 8. Yeast cells. DETAILED DESCRIPTION

[0030] Example 1

[0031] like Figure 1-5 As shown, an embodiment of the present invention provides a microfluidic chip, which includes a conductive cover plate 1 and a base plate 2, wherein a fluid inlet 5 and a fluid outlet 6 are provided above the cover plate, and an intermediate covering layer 3 for accommodating pine pollen microrobots and cell suspensions is provided between the cover plate 1 and the base plate 2, and electrodes 4 are respectively provided on the cover plate 1 and the base plate 2, and the electrodes 4 are connected to a signal generator for applying an alternating current signal through a wire.

[0032] In this embodiment, the cover plate 1 and the bottom plate 2 are both made of glass and are parallel and staggered and overlapped. The cover plate 1 and the bottom plate 2 are both coated with an indium tin oxide coating on the side in contact with the intermediate cover layer 3 .

[0033] In this embodiment, the electrode 3 is a metal copper film, which is adhered along the edge of the wide side of the non-overlapping portion of the cover plate 1 and the bottom plate 2 and is arranged toward the middle covering layer 3 .

[0034] In this embodiment, the length of the non-overlapping parts of the cover plate 1 and the bottom plate 2 is greater than the width of the metal copper film pasted on both sides, and the length of the metal copper film is less than the length and width of the cover plate 1 and the bottom plate 2.

[0035] In this embodiment, the intermediate covering layer 3 is a polyethylene terephthalate film with four surrounding grooves. The length and width of the polyethylene terephthalate film are smaller than the cover plate and the bottom plate. The PET film has good ductility and good adhesion to glass, and can directly contact the biological sample without causing adverse reactions.

[0036] Specifically, the glass is 30 mm long, 30 mm wide, and 1 mm high. Indium tin oxide is coated on the entire outer surface of one side of the glass with a thickness of 0.5 μm. The middle covering layer 3 is a PET film, which is shorter than the glass, 18 mm long, 28 mm wide, and 0.2 mm thick. Part of the middle layer is removed to open a 15 mm long and 24 mm wide groove. The metal copper film is 10 mm long and 4 mm wide, and is tightly attached to the edge of one side of the conductive glass with indium tin oxide (ITO) coating, and the connecting wires are led out. The radius of the inlet and outlet channels is 1 mm.

[0037] The preparation method of the microfluidic chip of the present invention comprises:

[0038] Clean the ITO glass with deionized water and wipe dry. Cut a PET film to the desired size, remove the protective film from one side of the film, and place it firmly against the center of the ITO-coated side of the glass, ensuring the bonding surface faces up, in a plasma bonder. Set the plasma power to 10 W and the oxygen flow to 8-10 ni / h for 32 seconds. Cut a portion of the PET film with a knife, create a groove around the center of the film, remove the protective film from the other half of the PET, and place another piece of glass, ITO-coated side down, against the PET film with a certain offset (ensuring that the PET film is covered by both layers of glass and that the glass itself is offset so that the edges extend beyond each other). Perform another plasma bond. Remove the film and let it rest for ten minutes, inspecting the edges of the grooves for bubbles (bubbles indicate unsuccessful bonding). Finally, transfer the film to a heated carbon plate, press down on the surface with a weight, and set the temperature to 80°C for two hours to obtain a bonded microfluidic chip.

[0039] Example 2

[0040] 1. Preparation of Sputtered Pine Pollen

[0041] 1) Mix 1g of pine pollen with 20ml of anhydrous ethanol and place in an ultrasonicator for 5-10 minutes. Filter the mixture with filter paper. Repeat the above operation three times for the obtained pine pollen. During this time, use a pipette to extract a small amount of sample and observe the removal of impurities and the dispersion of the pine pollen under a microscope.

[0042] 2) Mix the cleaned pine pollen with 20 ml of anhydrous ethanol and place it in an ultrasonicator for 5 minutes. Immediately use a pipette to extract 20 μl of the mixture and drop it onto a glass slide in small amounts several times. Use the flat plate streak method to spread the mixture as evenly as possible on the glass slide.

[0043] 3) Move the slides to a drying oven at 60°C for 20-30 minutes, then remove them and allow them to cool naturally to room temperature. Observe the dispersion of the pine pollen with the naked eye to avoid excessive stacking. Use tape to remove any clumps if necessary.

[0044] 4) Place the glass slide with pine pollen in the ion sputtering instrument. Use gold as the sputtering metal. Tighten the valve to extract the air in the sputtering chamber to a low vacuum environment (10 mmHg). Start sputtering for about 1-2 minutes. After sputtering is completed, adjust the direction of the glass slide and sputter again.

[0045] 5) Place the sputtered glass slide in a Petri dish and add an appropriate amount of anhydrous ethanol until the slide is completely immersed. Use a brush to gently scrape off the pine pollen (observe under a microscope: the originally transparent cytoplasm of the pine pollen is now covered with a dark green layer). Slowly introduce the mixture into a filter paper funnel, leaving the solids completely at the bottom of the funnel. Once all the anhydrous ethanol has dripped off, slowly drip deionized water onto the edge of the filter paper until the solids are submerged. Repeat this process 2-3 times to complete the cleaning process.

[0046] 2. Sample preparation:

[0047] In order to prevent cells from adhering to the glass substrate or edge PET during the experiment, thereby affecting experimental observation, an active agent solution can be prepared to inhibit cell adhesion.

[0048] Preparation of the active agent solution: Anhydrous ethanol and Tween solution were prepared in a volume ratio of 9:1 to obtain active agent solution A to prevent cell adhesion during the experiment; KCl was dissolved in deionized water and tested using a conductivity meter while stirring. The conductivity was adjusted to an appropriate conductivity (1-6mS / m) as cell buffer solution B to balance the osmotic pressure inside and outside the cells; then, solution A and solution B were mixed in a volume ratio of 1:99 to prepare solution C, which served as the cell culture medium for the experiment.

[0049] Mix 20 mg of dry yeast powder with 5 ml of deionized water, place in a sonicator for 5 minutes, and heat in a 50°C oven for two hours. Then, use a pipette to transfer 1 ml of the yeast suspension to a centrifuge tube. Centrifuge and repeat three times with Solution B to remove metabolic waste and impurities. Finally, place the washed yeast back into Solution C with the appropriate conductivity. Use a cell counting plate to adjust the concentration of the prepared solution.

[0050] 3. Experimental operation:

[0051] 1) Inject the prepared sputtered pine pollen 7 and the conductive yeast cell solution into the microfluidic chip through the fluid inlet 5 of the top ITO glass. Turn on the microscope, computer, and CCD camera in sequence. Place the copper film tightly against the extended edge of the ITO side of the two glass panels of the microfluidic chip (one for each of the upper and lower glass panels), without touching the PET film. Connect the wires and place the film on the stage of a fluorescence microscope. Adjust the objective lens as needed for the experiment, and secure the chip position and focal length.

[0052] 2) Connect the output port of the signal generator to the wire leading out of the chip, turn on the signal generator power supply, and after it stabilizes, adjust the voltage and frequency of the applied electrical signal (10-15V; 100-500Hz) to induce charge electroosmosis at the top of the sputtered pine pollen cavity, causing yeast cells to adsorb 8 and complete cell capture.

[0053] Figure 6-7 The unique structure of pine pollen is demonstrated. Its air sacs are hollow, so when it floats naturally in water, most of the time it presents an attitude with the air sacs facing upwards and the cytoplasm facing downwards. When sputtering occurs, metal substances will preferentially deposit on the top of the air sacs and on the partially exposed cytoplasm. These areas therefore have an electrical conductivity much higher than the surrounding environment.

[0054] Under the action of the AC electric field, the polarized dielectric particles (pine pollen microrobots) will generate induced charges under the action of the dipole moment, and the voltage will be concentrated in the place with larger conductivity, that is, the sputtered part of the pine pollen has a large amount of induced charge (similar to an electrode). At this time, the vertical electric field will be offset around the sputtered part, with the direction from the top of the airbag to the middle cytoplasm. Under the action of the non-uniform AC electric field, the current path will also be distorted, forming a local high current density area. These areas will produce a large amount of non-uniform temperature rise inside the solution due to the Joule effect of the current, thereby forming a temperature gradient, and the fluid temperature gradient The existence of causes the conductivity and dielectric constant in the solution to also form a conductivity gradient and dielectric gradient. Under the action of the AC electric field, the fluid flows along the gradient direction, which is the AC electrothermal coupling effect, generating a fluid vortex. At the same time, the target cells are also polarized by the electric field to generate induced charges, and they will move under the action of the non-uniform AC electric field, which is dielectrophoresis (DEP). The movement of the target cells is controlled by two forces, and the dominant force is different under different conditions. For example, larger yeast cells are affected by more dielectrophoretic force, and smaller yeast cells are affected by more fluid vortexes. In addition, fluid vortexes will only be generated when the voltage is concentrated at low frequency.

[0055] like Figure 8As shown, the red lines are electric field lines, and the color of the area represents the distribution of charge. At low frequencies, the charge is concentrated on both sides of the top of the air sac, which is the red and green area in the figure. This is also a necessary condition for the generation of fluid vortexes. The electric field lines inside the air sac are vertical, and the surrounding electric field is offset, resulting in a non-uniform electric field, especially in the middle and on both sides of the pollen cell. Figure 10 The direction of the fluid vortex force can be seen from the top of the air sac to the middle and both sides of the cell. The cell will move along Figure 10 The fluid moves in the direction of the vortex (blue line) and is eventually captured near the cell. Figure 9 As shown in the figure, at high frequencies, the charges are almost evenly distributed, the electric field will bypass the cells, and the direction is roughly the same. At this time, the fluid vortex is small or even disappears, and the cells are only attracted by the induced charges, and the capture of the cells is weakened.

[0056] This invention utilizes metal-sputtered pine pollen to design a microrobot that generates an induced electric potential, allowing it to directly capture specific target cells within a cell-medium solution. The overall microfluidic chip has a "sandwich" structure, with the top and bottom layers covered by conductive ITO glass. The middle layer is a working area framed by a PET film, and the top ITO glass layer features inflow and outflow ports for the cell-containing medium. When an alternating current signal is applied to the two ITO glass layers, induced charge electroosmotic flow (ICEO) is generated on the microrobot's surface within the cell-medium solution. Micro-vortices in the fluid guide cells near the two cavities of the pine pollen toward them. Furthermore, due to the electric field gradient, the target cells are subjected to dielectrophoretic (DEP) forces. Due to the unique geometric shape of the pine pollen, its nearly spherical air sac structure, very high local electric field strength is generated within the two cavities, accelerating the attraction of cells toward them. Compared with traditional cell capture solutions, this solution has low production costs, simple structure, and avoids the tedious microchannel design process; and the control process is completed by electronic control, which greatly reduces the probability of target cells being contaminated by the outside world.

Claims

1. An application of a pine pollen microrobot combined with a microfluidic chip in cell capture, characterized in that: The pine pollen microrobot is pine pollen sputtered with gold. The microfluidic chip is injected with the pine pollen microrobot and a cell solution with electrical conductivity. When an alternating current signal is applied to the microfluidic chip, the fluid around the pine pollen microrobot generates an induced charge electroosmotic vortex, causing the cells in the cell solution to move toward the cavity of the pine pollen microrobot, thereby achieving cell capture.

2. The application of the pine pollen microrobot combined with the microfluidic chip in cell capture according to claim 1, characterized in that: The cells in the cell solution move toward the cavity of the pine pollen microrobot under the action of dielectrophoretic force.

3. The use of the pine pollen microrobot combined with a microfluidic chip in cell capture according to claim 1, characterized in that: The pine pollen microrobot has two cavities with nearly spherical airbag structures. The nearly spherical airbag structures of the two cavities generate locally enhanced electric field intensity, thereby accelerating the movement of cells toward the cavities.

4. The use of the pine pollen microrobot combined with a microfluidic chip in cell capture according to claim 1, characterized in that: The voltage of the applied AC signal is 10-15V, and the frequency is 100-500HZ.

5. The use of the pine pollen microrobot combined with a microfluidic chip in cell capture according to claim 1, characterized in that: The cell solution with conductivity includes solution A and solution B, wherein solution A is an active agent solution prepared by mixing anhydrous ethanol and Tween solution in a volume ratio of 9:1, and solution B is a cell buffer solution with a conductivity of 1 to 6 mS / m prepared by dissolving potassium chloride in deionized water. The volume ratio of solution A to solution B is 1:

99.

6. The use of the pine pollen microrobot combined with a microfluidic chip in cell capture according to claim 1, characterized in that: The microfluidic chip includes a conductive cover and a base, a fluid inlet and a fluid outlet are provided above the cover, an intermediate covering layer for accommodating pine pollen microrobots and cell suspensions is provided between the cover and the base, and electrodes are respectively provided on the cover and the base, and the electrodes are connected to a signal generator for applying an alternating current signal through wires.

7. The use of the pine pollen microrobot combined with a microfluidic chip in cell capture according to claim 6, characterized in that: The cover plate and the bottom plate are both made of glass and are parallel and staggered and overlapped. The cover plate and the bottom plate are both coated with an indium tin oxide coating on the side in contact with the middle covering layer.

8. The use of the pine pollen microrobot combined with a microfluidic chip in cell capture according to claim 7, characterized in that: The electrode is a metal copper film, which is adhered along the edge of the wide side of the non-overlapping portion of the cover plate and the bottom plate and is arranged toward one side of the middle covering layer.

9. The use of the pine pollen microrobot combined with a microfluidic chip in cell capture according to claim 8, characterized in that: The lengths of the non-overlapping portions of the cover plate and the bottom plate are both greater than the widths of the metal copper films attached to both sides, and the lengths of the metal copper films are less than the widths of the cover plate and the bottom plate.

10. The use of the pine pollen microrobot combined with a microfluidic chip in cell capture according to claim 6, characterized in that: The middle covering layer is a polyethylene terephthalate film with four surrounding grooves. The length and width of the polyethylene terephthalate film are both smaller than those of the cover plate and the bottom plate.

Images