Photoelectric induction type self-driven microrobot and cell screening and carrying method

Abstract

The invention discloses a photoelectric induction type heterogeneous self-driven micro-robot and a cell screening and carrying method. Comprising a micro-robot body construction method and a cell screening and carrying method, wherein the cell screening and carrying method comprises three steps of a photoelectric field induced self-driving method, cell screening and cell carrying. According to the invention, the micro-robot body has heterogeneity, isomerism, smooth rotation appearance and self-driving capability. According to the method, the motion constraint of the micro-robot is formed through photoelectric field induction, and the motion direction is induced; in the aspect of cell screening, a distinguishable double-interaction mode is provided for cells with differentiated electrical properties based on three interaction functional areas of a micro-robot, and heterogeneous high-purity cell separation is realized; in the aspect of cell carrying, the micro-robot-cell interaction state is regulated and controlled by changing light patterns and alternating current electric field parameters, so that the micro-robot can load, transport and unload the cells according to needs. The micro-robot can realize integrated operation of cell screening, transportation, loading and unloading.

Description

Technical Field

[0001] The present invention belongs to the field of micro-nano processing technology and micro-nano robot biological operation, and specifically relates to a manufacturing method of a heterogeneous self-driving robot induced by a photoelectric field, a photoelectric field induced self-driving method, and a cell screening and carrying method. Background Art

[0002] Micro-nano manipulation technology at the single-cell level plays an increasingly important role in tissue engineering and biomedical engineering. At present, it has been confirmed that there are many methods for capturing, screening, transporting and other operations on single cells in microscale environments. Among them, methods such as building a cell movement model based on the dynamic interaction mechanism of multi-source physical fields and single cells, designing a cell screening scheme based on the analysis of the differentiated response mechanism of cells to physical fields, and realizing cell movement based on the dynamic modulation of physical fields in spatial regions provide effective ways for cell-level micro-nano biological manipulation tasks. However, the spatial continuity of the external physical field hinders the independent movement of single cells in the cell group, and the introduction of magnetic materials and high-power light energy will also affect the activity of cells. Therefore, this type of method cannot meet the urgent needs of tissue engineering and biomedical engineering research tasks for high flexibility and high biological affinity of cell micromanipulation methods.

[0003] In addition, the use of indirect agent strategies to achieve single-cell micromanipulation through microrobots similar to the cell scale is also common. For micrometer-scale cell manipulation tasks, microrobots are required to be able to screen, load, transport, and unload cells on demand. However, in the microscale environment, there are electrostatic forces, van der Waals forces, surface tension and other effects. Microrobots are either tightly attached to cells and cannot be separated, or they repel each other with cells, and the robustness of the interaction process with cells is extremely low. At the same time, the homogenized structure of the microrobot makes it difficult to stimulate differentiated motion responses of cells, and high-precision cell screening tasks cannot be achieved. In order to achieve the integrated operation tasks of cell screening, transportation, on-demand carrying and unloading, while meeting the requirements of high flexibility and high bioaffinity, a low-invasive microrobot that can move autonomously and interact with cells in a controllable manner is needed. Summary of the invention

[0004] The purpose of the present invention is to propose a photoelectrically induced heterogeneous self-driven microrobot and a cell screening and carrying method in order to realize the integrated functionalization of cell screening, transportation, loading and unloading of the microrobot, and at the same time to avoid the destruction of cell activity by factors such as physical fields and mechanical stress.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention proposes a photoelectrically induced heterogeneous self-driving microrobot and a cell screening and carrying method, including: a microrobot body structure, a cell screening and carrying method, wherein the cell screening and carrying method includes three steps: a photoelectric field induced self-driving method, cell screening and cell carrying; the present invention is characterized in that:

[0007] The microrobot body structure includes the smooth rotation appearance, heterogeneity, isomerism, and self-driving ability of the microrobot; in the photoelectric field induced self-driving part, the photoelectric field is used to form motion constraints and induce the motion direction, so as to realize the programmable and controllable motion of the microrobot; in terms of cell screening, based on the three major interactive functional areas of the microrobot, a distinguishable dual interactive mode is provided for cells with differentiated electrical characteristics, so as to realize the separation of heterogeneous high-purity cells; in terms of cell carrying, the parameters of the light pattern and the AC electric field are changed to regulate the microrobot-cell interaction state, so as to realize the microrobot's on-demand loading, transportation, and unloading operations of cells.

[0008] The microrobot body structure is characterized by the overall appearance, heterogeneity, heterogeneity, and self-driving ability of the microrobot. The microrobot adopts an arc-shaped smooth appearance and is a rotating body design, which prevents the regeneration of high-density local electric fields while ensuring the performance consistency of the microrobot during self-rotation. The top is an external convex structure, the middle is an annular concave structure, and the bottom is an internal concave structure; the microrobot body is a PDMS colloidal material and is manufactured by two-photon lithography; the upper part of the microrobot is covered with a thin film composed of 10nm chromium and 150nm gold, and the metal film is prepared by electron beam evaporation; PMMA is used as a sacrificial layer to protect the lower part of the microrobot from being coated, ensuring that the metal film coverage area does not exceed 65% of the surface area of ​​the microrobot and is not less than 35% of the surface area, forming a heterogeneous and heterogeneous microrobot body; the microrobot can produce spontaneous movement behavior under the influence of the unbalanced flow of charged particles on both sides in an AC electric field environment, that is, it has self-driving ability.

[0009] The microrobot photoelectric field induced self-driving step is characterized by the motion constraint of the microrobot by the photoinduced dielectrophoretic force, the induction of the self-driving direction by the photoinduced electric field, and the switching of the self-driving direction. The microrobot has the ability to self-drive in the AC electric field, but the direction of movement is uncontrollable and the motion trajectory is random. The programmable light pattern generated by the DMD projector is focused to the light-induced chip after passing through a convex lens, a short-pass filter, a beam splitter, and an objective lens, and a local non-uniform electric field is formed under the action of the AC electric field generated by the signal generator. In the local non-uniform electric field, the microrobot is attracted to the edge of the light pattern by the positive dielectrophoretic force, forming a motion constraint perpendicular to the edge of the light pattern. The colloid-metal interface of the microrobot is perpendicular to the edge of the light pattern under the action of the electric field, causing the microrobot to self-drive along the edge of the light pattern. Under the low-frequency electric field, the self-driving behavior of the microrobot is dominated by the AC charge electrophoresis, and the movement is toward the colloid side; under the high-frequency electric field, the self-driving behavior is dominated by the self-dielectrophoresis, and the movement is toward the metal film side.

[0010] The microrobot cell screening step is characterized in that there are two distinguishable and observable differentiated interaction modes between the microrobot and the cells. In general, the cells and the microrobots subjected to the positive dielectrophoretic force repel each other, and at the same time maintain a relatively close distance under the action of the light pattern induced electric field, so that the cells are located at the front end of the protrusion 1 on the top of the microrobot, which is counted as interaction mode I; the cells and the microrobots subjected to the negative dielectrophoretic force attract each other, and the cells are adsorbed on the bottom depression 4 or the middle depression 2 of the microrobot, which is counted as interaction mode II. The membrane capacitance and cell size of different types of cells are inconsistent, so they have differentiated electrical properties. By selecting an appropriate alternating current frequency, different types of cells in the same environment show different interaction modes with the microrobot. The difference between the two interaction modes in microscopic observation is very significant, which can be used as a basis for judging the cell category, combined with the cell carrying method of the microrobot to achieve the screening of target cells.

[0011] The microrobot cell carrying step is characterized by three parts: cell loading, transportation and unloading under dynamic modulation of light patterns and AC electric field parameters. Cell loading is achieved when the microrobot and the cell complete interaction in the electric field. If the cell and the microrobot present interaction mode I, the microrobot uses the mutual repulsive force to push the cell toward the target position to achieve cell carrying. The width of the light pattern is adjusted to destroy the equilibrium state of interaction mode I to achieve cell unloading. If the width is increased, the microrobot bypasses the cell; if the width of the light pattern is reduced, the microrobot returns to the original path. If the cell and the microrobot present interaction mode II, the microrobot uses attraction to lead the cell toward the target position to achieve cell carrying. Reduce the frequency of the alternating current, the polarity of the cell charge is deflected, the equilibrium state of interaction mode II is destroyed, the cell and the microrobot are separated, and the microrobot moves toward the colloid side to return to the original path to achieve cell unloading. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The drawings described herein are used to provide a further understanding of the embodiments of the present invention, constitute a part of this application, and do not constitute a limitation of the embodiments of the present invention. In the drawings:

[0013] Figure 1 This is a schematic diagram of the single-cell screening and transporting principle of the heterogeneous self-driven microrobot of the present invention;

[0014] Figure 2 This is an optical principle diagram of the microrobot's self-driving induced by the photoelectric field in the present invention;

[0015] Figure 3 Schematic diagram of microrobot cell transport based on interactive mode I in the present invention;

[0016] Figure 4 Schematic diagram of microrobot cell transport based on interactive mode II in the present invention;

[0017] Figure 5 Schematic diagram of microrobot cell unloading based on interactive mode I in the present invention;

[0018] Figure 6 Schematic diagram of microrobot cell unloading based on interactive mode II in the present invention;

[0019] The meanings of the various reference numerals are as follows: 1-chromium / gold film, 2-depression in the middle of the microrobot, 3-PDMS microrobot body, 4-bottom depression, 5-DMD projector, 6-LED light, 7-computer, 7-CCD camera, 9-long pass filter, 10-beam splitter, 11-convex lens, 12-short pass filter, 13-objective lens, 14-light-induced dielectrophoresis chip, 15-signal generator, 16-light pattern, 17-carrier cell, 18-microrobot DETAILED DESCRIPTION

[0020] The present invention is further described with reference to the following examples, but the examples do not constitute any limitation to the present invention.

[0021] (1) Micro robot body construction, the specific process is as follows:

[0022] S1: Print the microrobot body on the substrate using a two-photon lithography device, the material is polydimethylsiloxane (PDMS);

[0023] S2: Spin-coat polymethyl methacrylate (PMMA) on the substrate carrying the PDMS body using a spin coater to a specified thickness, which is about half the height of the microrobot body and covers the lower half of the microrobot;

[0024] S3: Under a pressure lower than 〖2×10〗^(-6) Pascal, a 10nm thick chromium film is first evaporated using an electron beam evaporation device as a connecting material; then a 150nm thick gold film is deposited.

[0025] S4: Use acetone to dissolve the PMMA on the surface of the substrate, and use clean water to clean the substrate. Repeat this operation three times.

[0026] S5: The microrobot body is obtained, the upper part of which is covered with a metal film and the lower part is a bare PDMS body.

[0027] S6: Add an appropriate amount of clean water, use ultrasound to separate the microrobot from the substrate, remove the supernatant after centrifugation, and retain the suspension containing a high concentration of microrobots.

[0028] (2) The microrobot performs cell screening operations. The specific process is as follows:

[0029] S1: Detect the cell membrane capacitance of the target cell type and the impurity cell type, and calculate the crossover frequency of each cell, and calculate the average crossover frequency between the target cell and each impurity cell, recorded as f1, f2, ..., fn, where n is the number of impurity cell types;

[0030] S2: releasing the self-driving microrobot described above in the dielectrophoresis chip, generating a light pattern path connecting the microrobot and the target cell through the DMD projector 5, and inducing the microrobot to move to the rear of the cell to be screened;

[0031] S3: reducing the output amplitude of the AC signal of the AC signal generator 15 to 0, and setting the frequency of the AC signal to f1, f2, ..., fn in sequence, and executing the operations of S4 to S6 after each setting;

[0032] S4: increasing the AC signal output amplitude of the AC signal generator 15 to 10 Vpp, and observing the interaction mode between the cell and the microrobot;

[0033] S5: If the cell and the microrobot repel each other, and the cell is located at the front end of the protrusion 1 on the top of the microrobot, it is interaction mode I. If the cell and the microrobot attract each other, and the cell is attached to the bottom depression 4 or the middle depression 2 of the microrobot, it is interaction mode II;

[0034] S6: Determine the crossover frequency of the cells to be screened based on the different interaction modes between the cells and the microrobot. If the current cell to be detected can be determined as an impurity cell, execute S2 to detect the remaining cells to be screened. If the current cell to be detected can be determined as a target cell, execute S7;

[0035] S7: Use the single cell delivery method to transport the target cells to the cell collection area and unload them to complete the screening of target cells

[0036] (3) The microrobot performs cell transport operations. The specific process is as follows:

[0037] S1: releasing the self-driving microrobot in the dielectrophoresis chip, generating a light pattern path connecting the microrobot and the target cell through the DMD projector 5, and inducing the microrobot to move to the cell to be carried;

[0038] S2: Wait for the microrobot to interact with the carrier cell and generate a light pattern path connecting the microrobot and the target location;

[0039] S3: Observe the microrobot-cell interaction mode. If it is interaction mode I, adjust the width of the light pattern to ensure that the target cell moves along the light pattern under the contactless push of the microrobot; if it is interaction mode II, execute step S6;

[0040] S4: transport the target cells to the target location,

[0041] S5: Adjust the width of the light pattern to achieve unloading of the target cell. If the microrobot needs to bypass the cell, increase the width of the light pattern; if the microrobot needs to return to the original path, reduce the width of the light pattern;

[0042] S6: If the microrobot and the target cell show interaction mode II, adjust the frequency of the AC signal to 100kHz, and increase the width of the light pattern to increase the carrying speed. The microrobot uses the middle depression and the bottom depression to carry up to 3 cells at the same time, and the cells are separated from each other.

[0043] S7: Arriving at the target position, the frequency of the AC signal is adjusted to 10kHz, the target cell and the microrobot are separated from each other, and the microrobot reverses its movement direction and returns to the original path to achieve unloading of the target cell.

[0044] The above is only a specific embodiment of the present invention, which is only used to illustrate the technical solution and inventive concept of the present invention and does not limit the scope of the claims of the present invention. Any other technical solutions that can be obtained by technicians in this technical field based on the inventive concept of this patent and in combination with the prior art through logical analysis, reasoning or limited experiments should also be considered to fall within the scope of protection of the claims of the present invention.

Claims

1. A photoelectrically induced self-driven microrobot and a cell screening and transport method, comprising: The microrobot body structure and the cell carrying and screening method, wherein the microrobot cell carrying and screening includes three steps: photoelectric field induced self-driving, cell screening and cell carrying; the microrobot structure and the method of using the microrobot are characterized by: The microrobot body structure includes the smooth rotation appearance, heterogeneity, isomerism, and self-driving ability of the microrobot; in the photoelectric field induced self-driving step, the photoelectric field is used to form motion constraints and induce the motion direction, so as to realize the programmable and controllable motion of the microrobot; then in the cell screening step, based on the three major interactive functional areas of the microrobot, a distinguishable dual interactive mode is provided for cells with differentiated electrical characteristics, so as to realize the separation of heterogeneous high-purity cells; in the cell carrying step, the parameters of the light pattern and the AC electric field are changed to regulate the microrobot-cell interaction state, so as to realize the on-demand loading, transportation, and unloading operations of the microrobot on cells. The characteristics of each part are: As for the microrobot's main structure, the microrobot adopts a smooth rotating appearance design; there are depressions in the middle and bottom to form interactive functional areas; the main body of the microrobot is PDMS colloid, and the upper part is covered with a thin film composed of 10nm chromium and 150nm gold; the microrobot has self-driving ability in an AC electric field. The cell screening and delivery method section: In the microrobot photoelectric field induced self-driving step, the light-induced electric field generates motion constraints on the microrobot and induces its self-driving direction. The switching of the self-driving direction can be achieved by changing the electric field frequency. The microrobot cell screening step utilizes two distinguishable and observable differentiated interaction modes between the microrobot and cells, combined with a cell transport method, to achieve cell screening. The microrobot cell carrying step achieves loading, transporting and unloading of cells subjected to positive dielectrophoretic force by modulating the width of the light pattern, and achieves loading, transporting and unloading of cells subjected to negative dielectrophoretic force by modulating the frequency of the electric field.

2. The photoelectrically induced self-driven microrobot and cell screening and transport method according to claim 1, characterized in that: The micro robot body structure further includes: The microrobot has a smooth arc appearance and a rotating body design, which prevents the regeneration of high-density local electric fields while ensuring the performance consistency of the microrobot during self-rotation. The bottom is an inner concave structure, the top is an outer convex structure, and the middle is an annular concave structure; the main body of the microrobot is PDMS colloid material; the upper part of the microrobot is covered with a thin film composed of 10nm chromium and 150nm gold. It is necessary to ensure that the metal film coverage area does not exceed 65% of the surface area of ​​the microrobot and is not less than 35% of the surface area, forming a heterogeneous and heterogeneous microrobot body; the microrobot can produce spontaneous movement behavior under the influence of the unbalanced flow of charged particles on both sides in an AC electric field environment, that is, it has self-driving ability.

3. The photoelectrically induced self-driven microrobot and cell screening and transport method according to claim 1, characterized in that: The microrobot cell screening step further comprises: S1: Turn on the AC signal, and the microrobot interacts with the cells to be screened in the electric field. S2: Measure the membrane capacitance of all cells to be tested by type and calculate their crossover frequency. S3: Observe and determine the interaction mode between the microrobot and the cell: if the cell and the microrobot repel each other and the cell is located at the front end of the protrusion 1 on the top of the microrobot, it is counted as interaction mode I; if the cell and the microrobot attract each other and the cell is adsorbed on the bottom depression 4 or the middle depression 2 of the microrobot, it is counted as interaction mode II. S4: If it is interactive mode I, it is determined that the crossover frequency of the cells to be detected is higher than the current electric field frequency; if it is interactive mode II, it is determined that the crossover frequency is lower than the current electric field frequency. S5: Continuously changing the frequency of the electric field, and analyzing the crossover frequency range of the cells to be detected according to the interaction mode, until the type of the cells to be detected is obtained through analysis. S6: Combined with the cell delivery step, the target cells can be screened and purified.

4. The photoelectrically induced self-driven microrobot and cell screening and transport method according to claim 1, characterized in that: The microrobot cell carrying step further comprises: S1: The microrobot and cells complete interaction in the electric field to achieve cell loading. S2: If the interaction mode between the cell and the microrobot is I, the microrobot uses the mutual repulsive force to push the cell toward the target position to achieve cell transportation. If the interaction mode between the cell and the microrobot is II, execute S4. S3: Adjust the width of the light cluster to destroy the equilibrium state of interaction mode I and realize cell unloading. If the width is increased, the microrobot will bypass the cell; if the width of the light cluster is reduced, the microrobot will return to the original path. S4: If the interaction mode between the cell and the microrobot is II, the microrobot carries the cell toward the target location through attraction. S5: The frequency of alternating current is reduced, the polarity of cell charge is deflected, the equilibrium state of interaction mode II is destroyed, the cell is separated from the microrobot, and the microrobot moves toward the colloid side and returns to the original path to achieve cell unloading.

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