A method for regulating the transmission path of an intravascular target object in a living animal based on an optical tweezer

Abstract

The application provides a method for regulating transmission path of target objects in blood vessels of living animals based on optical tweezers, and belongs to the technical field of photoelectricity. The application constructs a biological micro-router in the blood vessels of living animals by simultaneously capturing five red blood cells and arranging them into an equilateral pentagon; a ring-shaped scanning optical potential well is applied to each captured red blood cell, and then the red blood cell is driven to rotate along a specific axis under the action of optical torque, and the speed and direction of rotation can be real-time regulated. Each rotating red blood cell can be regarded as an endogenous cell micro-rotor. Under the driving of the five cell micro-rotors, a specific driving micro-flow field can be generated in the surrounding area of the target objects. At this time, the target objects in the micro-flow field will flow synchronously under the action of fluid viscous force, thereby realizing the targeted delivery of the target objects. The application can realize the dynamic input, internal processing and controllable output of the target objects, thereby completing the dynamic routing selection of the target objects.

Description

Technical Field

[0001] This invention belongs to the field of optoelectronic technology, specifically relating to a method for controlling the transport path of a target within the blood vessels of a living animal based on optical tweezers. Background Technology

[0002] Achieving dynamic routing of different blood components within living blood vessels—that is, guiding their directional migration along their respective predetermined paths—is of great significance in a range of biomedical applications, including cell separation, drug delivery, medical diagnostics, immunotherapy, and clinical surgery. Inspired by the cooperative behaviors of the biological world, such as those of birds and insect colonies, programmable medical micromachines (PMMs) possess advantages such as small size (micrometer size), active propulsion, and wireless remote control, making them potentially valuable for trajectory control of different targets and subsequent routing. Furthermore, thanks to the high reconfigurability and swarm intelligence of PMMs, the interactions between different building blocks can be precisely controlled according to specific environmental conditions, providing excellent programming and adjustment capabilities to execute desired target tasks. Over the past few decades, researchers have developed PMMs suitable for dynamic routing of various targets by integrating micro / nanofabrication techniques and external field-induced interactions. For example, by setting the rotation frequency of an external magnetic field lower than the step frequency of selected artificial bacterial flagella (ABFs) but higher than the step frequency of unselected ABFs, dynamic routing of two artificial bacterial flagella towards different branches was successfully achieved.

[0003] However, most of the aforementioned PMMs are based on synthetic materials. Due to biocompatibility or biodegradability issues, they are generally difficult to apply in in vivo studies. Recently, many biological cells, such as E. coli, microalgae, and yeast cells, have been shown to be used as natural building blocks for assembling PMMs, thus bridging the gap between synthetic machinery and the biological world. However, since they are not endogenous materials naturally present in the body, they require invasive implantation to introduce them into the body, which may trigger unwanted immune responses.

[0004] How to assemble naturally biocompatible PMMs based on endogenous cells, and thus achieve precise routing and selection of different blood components in the body, remains to be solved. Summary of the Invention

[0005] The purpose of this invention is to provide a method for controlling the transport path of a target in the blood vessels of a living animal based on optical tweezers. The method of this invention is based on red blood cells in the blood vessels and can achieve precise routing selection of different blood components.

[0006] This invention provides a method for constructing intravascular biological microrouters in living animals, comprising the following steps:

[0007] Red blood cells are captured and arranged into biological microrouters using an optical potential trap; the shape of the biological microrouters is an equilateral pentagon; each biological microrouter consists of 5 red blood cells;

[0008] Each red blood cell, as a cellular microrotor, is located at the vertex of an equilateral pentagon; the gaps between adjacent cellular microrotors in each of the biological microrouters constitute ports;

[0009] A ring-shaped scanning optical potential trap is applied to each of the cell microrotors;

[0010] When the laser focus is scanned along the circular trajectory, the cell microrotor rotates; the direction of the circular trajectory scanning laser focus is the same as the direction of the cell microrotor's rotation.

[0011] In the biological micro-router, in a counterclockwise direction, the cell micro-rotor adjacent upstream of the port is used to control the port to be in an input state or a closed state, and the cell micro-rotor adjacent downstream of the port is used to control the port to be in an output state or a closed state.

[0012] With all ports closed, the rotation direction of each cell micro-rotor in the biological micro-router is either clockwise or counterclockwise.

[0013] Preferably, the rotation speed of the cell microrotor is 1 to 18 rad / s.

[0014] Preferably, the optical potential well for capturing red blood cells is a fixed optical potential well; the fixed optical potential well captures the center position of the red blood cells; the annular scanning optical potential well is centered on the center of the red blood cells; and the diameter of the annular scanning optical potential well is less than or equal to the diameter of the red blood cells.

[0015] Preferably, each of the annular scanning optical potential wells comprises 3000 optical potential wells.

[0016] Preferably, the scanning frequency of the annular scanning optical potential well is ≤100000Hz.

[0017] Preferably, the side length of the biological microrouter is the distance between the center points of adjacent cell microrotors; the ratio of the biological router to the blood vessel diameter is ≤0.75.

[0018] The present invention also provides a live animal intravascular biological microrouter constructed by the method described above.

[0019] This invention also provides a method for regulating the transmission path of target objects within the blood vessels of live animals based on the above-described scheme, comprising the following steps:

[0020] The method includes regulating the input of the target analyte into the biological microrouter and / or regulating the output of the target analyte from the biological microrouter;

[0021] The regulation of target input to the biological micro-router includes: with all ports closed, adjusting the direction of the circular trajectory scanning laser focus and reversing the rotation direction of the cell micro-rotor adjacent upstream of the port to make the port an input state, and the target is input into the biological micro-router from this port;

[0022] The output of the target substance from the biological micro-router includes: with all ports closed, adjusting the direction of the circular trajectory scanning laser focus and reversing the rotation direction of the adjacent cell micro-rotors downstream of the port to make the port in an output state, and the target substance is output from the biological micro-router.

[0023] The method described is not for therapeutic purposes.

[0024] Preferably, after the target object is input into the biological micro-router, the method further includes: adjusting the rotation direction of the cell micro-rotor adjacent to the upstream of the port so that the port is in a closed state;

[0025] After the target object is output to the biological micro-router, the method further includes: adjusting the rotation direction of the adjacent cell micro-rotor downstream of the port so that the port is in a closed state.

[0026] Preferably, the method further includes adjusting the rotation radius of the target object within the biological micro-router;

[0027] The method for controlling the rotation radius of the target object within the bio-microrouter includes: adjusting the direction of the circular trajectory scanning laser focus, reversing the rotation direction of any one cell microrotor, and setting one port to an output state, causing the rotating target object to undergo linear transport to the outside of the bio-microrouter; before the target object is output from the bio-microrouter, adjusting the port controlled by the cell microrotor to a closed state, causing the target object to rotate within the bio-microrouter with a larger radius; or, by moving the target object towards the center of the bio-microrouter, causing the target object to rotate within the bio-microrouter with a smaller radius.

[0028] This invention provides a method for controlling the delivery path of target substances within the blood vessels of living animals based on optical tweezers. This method organically combines endogenous erythrocytes from living animals, scanning optical tweezers (SOTs), and an optical flow manipulation strategy. By simultaneously capturing five erythrocytes and arranging them into an equilateral pentagon, a biological micro-router is constructed within the blood vessels of the living animal. Furthermore, this invention applies a ring-shaped scanning optical potential trap to each captured erythrocyte, thereby driving the erythrocyte to rotate along a specific axis under the action of optical torque, and the speed and direction of rotation can be controlled in real time. Each rotating erythrocyte can be considered as an endogenous cellular microrotor. Driven by the five cellular microrotors, a specific driving microfluidic field is generated around the target substance. At this time, the target substance within the microfluidic field will flow synchronously under the action of fluid viscosity, thereby achieving targeted delivery of the target substance. Based on this, by controlling the speed and direction of the flow field at different locations in real time, dynamic input, internal processing, and controllable output of different target substances (such as platelets, leukocytes, and nanomedicines) can be achieved, thereby completing their dynamic routing selection. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0030] Figure 1 A schematic diagram illustrating the routing selection for different targets using an endogenous erythrocyte micro-router.

[0031] Figure 2 This is a schematic diagram of the device.

[0032] Figure 3This is a schematic diagram of the flexible assembly of a biological micro-router in vivo; (a1-a4) rotating a red blood cell clockwise; (a5-a8) rotating a red blood cell counterclockwise; (b1) probability distribution of the rotation radius of the red blood cell; (b2) probability distribution of the rotation angular velocity of the red blood cell; (b3) graph of the rotation angular velocity of the red blood cell over time; (c) rotating two red blood cell micro-rotors clockwise with a period of 0.8s; (d1) arranging three red blood cell micro-rotors in a straight line and rotating clockwise; (d2) arranging three red blood cell micro-rotors in a triangle and rotating clockwise; (e1) arranging four red blood cell micro-rotors in a straight line and rotating clockwise; (e2) arranging four red blood cell micro-rotors in a rectangle and rotating clockwise; (f) arranging four red blood cell micro-rotors in a pentagon to serve as a red blood cell micro-router; (f1-f3) all micro-rotors rotate clockwise; (f4-f5) changing the rotation direction and speed of micro-rotor 1.

[0033] Figure 4 The diagram illustrates three functions of a biological micro-router: (a1) signal input function of a macro-router; (a2) dynamic input function of a target object of a red blood cell micro-router; (b) input of a target object from port 3; (c1) signal output function of a macro-router; (c2) controllable output function of a target object of a red blood cell micro-router; (d) output of a target object from port 5; (e1) signal processing function of a macro-router; (e2-e3) internal processing function of a red blood cell micro-router for multiple targets; (f1) rotating a single target object counterclockwise; (f2) rotating a single target object clockwise; (g) increasing the rotation radius of a single target object rotating counterclockwise; (h) decreasing the rotation radius of a single target object rotating counterclockwise; (i) rotating two close targets counterclockwise; (j) rotating two separated targets counterclockwise; (k1) transporting two targets to the right; (k2) transporting two targets to the left; (k3) separating two targets; (l) rotating one target object while keeping the other stationary.

[0034] Figure 5 The diagram illustrates the dynamic routing capability of a biological micro-router. (a1) A schematic diagram of a target object inputting along the same port and outputting along different ports; (a2-a6) A target object inputting along port 2 and outputting along port 1; (a7) A target object inputting along port 2 and outputting along port 3; (a8) A target object inputting along port 2 and outputting along port 4; (a9) A target object inputting along port 2 and outputting along port 5; (b) Three target objects inputting from different ports and outputting from the same port; (c) Three target objects inputting from the same port and outputting from the same port; (d) Three target objects inputting from different ports and outputting from different ports; (e) Platelets inputting along port 2 and outputting along port 4; (f) White blood cells inputting along port 2 and outputting along port 4.

[0035] Figure 6 This is a biomedical application of a bio-microrouter; (a) a schematic diagram of using a red blood cell microrouter to guide platelets to control hemostasis, transport white blood cells for targeted clearance, and actively deliver antithrombotic nanomedicines; (b) delivering four platelets to a damaged blood vessel; (c) delivering platelets and white blood cells to damaged blood vessels and cell debris, respectively; (d) delivering nanomedicines to branch I; (e) delivering a group of nanomedicines to a specific thrombus. Detailed Implementation

[0036] This invention provides a method for constructing intravascular biological microrouters in living animals, comprising the following steps:

[0037] Red blood cells are captured and arranged into biological microrouters using an optical potential trap; the shape of the biological microrouters is an equilateral pentagon; each biological microrouter consists of 5 red blood cells;

[0038] Each red blood cell, as a cellular microrotor, is located at the vertex of an equilateral pentagon; the gaps between adjacent cellular microrotors in each of the biological microrouters constitute ports;

[0039] A ring-shaped scanning optical potential trap is applied to each of the cell microrotors;

[0040] When the laser focus is scanned along the circular trajectory, the cell microrotor rotates; the direction of the circular trajectory scanning laser focus is the same as the direction of the cell microrotor's rotation.

[0041] In the biological micro-router, in a counterclockwise direction, the cell micro-rotor adjacent upstream of the port is used to control the port to be in an input state or a closed state, and the cell micro-rotor adjacent downstream of the port is used to control the port to be in an output state or a closed state.

[0042] With all ports closed, the rotation direction of each cell micro-rotor in the biological micro-router is either clockwise or counterclockwise.

[0043] In this invention, the device used in the method is constructed around the Tweez250si scanning optical tweezers system (SOT) and combined with an inverted optical microscope for real-time observation and recording. In specific implementations of this invention, the device used in the method is preferably the device for regulating erythrocytes within the blood vessels of a living animal disclosed in patent CN202010582545.3.

[0044] In this invention, the number of the biological microrouters is one or more. Preferably, the side length of the biological microrouter is the distance between the center points of adjacent cell microrotors; the ratio of the biological router to the blood vessel diameter is preferably ≤0.75 to ensure stable assembly of the router within the blood vessel; the living animal blood vessel is measured in terms of the main artery or vein of a zebrafish, and the side length of the biological microrouter is preferably 10.5–17.9 μm.

[0045] In this invention, the optical potential well for capturing red blood cells is preferably a fixed optical potential well; the fixed optical potential well is set by focusing a specified laser beam onto the irradiance position of the red blood cells; the fixed optical potential well is preferably located at the center of the red blood cells. In this invention, the wavelength of the capturing laser beam forming the fixed optical potential well is preferably 1064 nm, which is within the biosafety window and can achieve a large penetration depth without causing additional photothermal damage; the capturing laser beam is preferably a continuous Gaussian beam; the power of the capturing laser forming the fixed optical potential well is preferably 50-100 mW.

[0046] In this invention, five fixed optical potential traps are used to capture five red blood cells in the blood vessels of a live animal and arrange the five red blood cells into an equilateral pentagon to form a biological micro-router; preferably, the five red blood cells are captured one by one; after being captured, the five red blood cells are located in the same plane.

[0047] The reason for using red blood cells to construct the bio-microrouter in this invention is that, as the most abundant blood cells in the circulatory system, red blood cells possess advantages such as excellent biocompatibility, long circulation, and non-immunogenicity, making them ideal candidates for constructing endogenous, programmable medical micromachines. Furthermore, the biological movement of red blood cells can be precisely controlled through external fields; for example, optical forces with single-cell precision can achieve precise manipulation of red blood cell movement in a non-contact and non-invasive manner. The bio-microrouter of this invention is entirely based on in vivo biological cells and combines the inherent advantages of scanning optical tweezers and optofluidic technology. By utilizing endogenous red blood cells, the constructed bio-microrouter avoids the invasive implantation of exogenous synthetic materials, exhibits high biocompatibility, and causes no invasive tissue damage or unnecessary immune responses.

[0048] The reason for setting the shape of the biological router to an equilateral pentagon in this invention is to break the inherent symmetry in order to achieve independent and simultaneous control of multiple targets; in addition, it is difficult to assemble a biological router using 7 red blood cells due to the size limitation of blood vessel diameter.

[0049] In this invention, the direction of the laser focus for the annular trajectory scanning is the same as the rotation direction of the cell microrotor. Scanning the laser focus clockwise along the annular trajectory of the cell microrotor causes the cell microrotor to rotate clockwise, and scanning the laser focus counterclockwise along the annular trajectory causes the cell microrotor to rotate counterclockwise. This controlled rotation exhibits high stability. By changing the scanning direction of the laser focus on the annular trajectory of the cell microrotor, this invention alters the input or output state of the controlled port, enabling the target object to be input into the biological micro-router from a specific port and output from a specific port of the biological micro-router, thus achieving the transmission of the target object along a predetermined path.

[0050] In this invention, within the biological micro-router, five red blood cells are sequentially designated as the first, second, third, fourth, and fifth cell microrotors in a counter-clockwise direction. The designations "first," "second," etc., are not intended to limit the actual function of the micro-router but are merely for descriptive convenience.

[0051] In this invention, the gap between the first and second cell microrotors constitutes port one; the gap between the second and third cell microrotors constitutes port two; the gap between the third and fourth cell microrotors constitutes port three; the gap between the fourth and fifth cell microrotors constitutes port four; and the gap between the fifth and first cell microrotors constitutes port five. The first, second, third, fourth, and fifth cell microrotors are used to control port one, port two, port three, port four, and port five to be in an input state or a closed state, respectively. The first, second, third, fourth, and fifth cell microrotors are used to control port five, port one, port two, port three, and port four to be in an output state or a closed state, respectively.

[0052] In this invention, taking the first cell micro-rotor as an example, when all other cell micro-rotors rotate clockwise, scanning the laser focus clockwise along the circular trajectory of the first cell micro-rotor causes the first cell micro-rotor to rotate clockwise, with ports one and five in a closed state, preventing the target object from being input into or output from the biological micro-router. When scanning the laser focus counterclockwise along the circular trajectory of the first cell micro-rotor, the first cell micro-rotor rotates counterclockwise, with port one in an input state and port five in an output state, allowing the target object outside the biological micro-router to be input into the biological micro-router through port one, and the target object inside the biological micro-router to be output from the biological micro-router through port five.

[0053] In this invention, the annular scanning optical potential well is a circular scanning optical potential well. Preferably, the annular scanning optical potential well is centered on the center of the red blood cell; preferably, the diameter of the annular scanning optical potential well is less than or equal to the diameter of the red blood cell.

[0054] In this invention, five red blood cells are arranged and rotated independently and programmably by scanning a laser focus along a circular trajectory in a time-division manner, constructing a biological micro-router at a designated location, thereby obtaining a microfluidic field with adjustable speed and direction. This invention utilizes an induced microfluidic-driven photofluidic strategy to achieve dynamic routing selection for various targets with single-cell precision and programmability.

[0055] In this invention, each of the annular scanning optical potential wells preferably includes 3000 optical potential wells, which can ensure that the cell microrotor rotates at maximum speed.

[0056] In this invention, the scanning frequency of the annular scanning optical potential well is preferably ≤100000Hz, more preferably 10000Hz, 20000Hz, 30000Hz, 40000Hz, 50000Hz, 60000Hz, 70000Hz, or 80000Hz, and most preferably 60000Hz; the power of the scanning laser forming the annular scanning optical potential well is preferably 50-100mW; the rotation period of the cell microrotor is preferably 0.8-1.2s, more preferably 0.9s. In this invention, the rotation speed of the cell microrotor is preferably 1-18rad / s, more preferably 7-8rad / s; in this invention, the rotation speed of the cell microrotor is affected by the number of potential wells contained in the annular scanning optical potential well, the scanning frequency, and the laser power. In the specific implementation of this invention, the rotation speed of the cell microrotor is controlled by changing the laser power; the rotation radius of the cell microrotor is preferably 5.8μm.

[0057] In one embodiment of the present invention, the scanning frequency of the annular scanning optical potential well is 100,000 Hz, and each annular scanning optical potential well has 3,000 optical potential wells. The laser will then scan cyclically according to the sequence: 1 / 2 / 3…2998 / 2998 / 3000—1 / 2 / 3…2998 / 2999 / 3000—1 / 2 / 3…2998 / 2999 / 3000… In this way, within 1 second, the laser will irradiate each optical potential well approximately 33 times, achieving a quasi-static distribution of the laser across the 3,000 optical potential wells. When a ring-shaped potential trap is applied to a red blood cell that is captured by a single potential trap, the center of the ring-shaped potential trap is made to coincide with the single potential trap. This causes the red blood cell to rotate around the position of the single potential trap. One end of the red blood cell is captured by the ring-shaped potential trap. Since the ring-shaped potential trap scans repeatedly in a circular pattern, one end of the red blood cell rotates along the scanning sequence of the ring-shaped potential trap, thus achieving the rotation of the red blood cell.

[0058] At this point, each optical potential well can be considered as being continuously illuminated by a laser beam, thus each optical potential well can stably capture one red blood cell. Therefore, by setting up five optical potential wells, five red blood cells can be captured simultaneously. Moreover, the position of each optical potential well can be independently manipulated, thus enabling the independent manipulation of specific red blood cells by moving specific optical potential wells while keeping the positions of other optical potential wells unchanged. This allows for the capture and arrangement of five red blood cells.

[0059] In the specific implementation of this invention, MATLAB software is used to merge the position coordinates of multiple optical potential wells in a ring-shaped scanning optical potential well into a patterned scanning sequence. The laser then moves along the sequence in a predetermined order under the action of an acousto-optic deflector, thereby enabling multifunctional operation of the red blood cells. The center position of the scanning trajectory corresponding to the scanning sequence can be moved in real-time with the mouse as needed. Placing this scanning sequence onto a red blood cell already captured by a single optical potential well causes the red blood cell to rotate as the laser dynamically scans along the sequence. By setting the optical potential wells in the sequence in the opposite order, the red blood cell rotates in the opposite direction. Furthermore, by placing five circular optical potential wells onto five red blood cells already captured and arranged in a pentagon, the assembly of a red blood cell micro-router is achieved.

[0060] In this invention, the plane of rotation of the cell microrotor is parallel to the blood flow trajectory. In this invention, the rotational speed of the cell microrotor affects the transport speed of the target substance; the faster the rotational speed of the cell microrotor, the faster the transport speed of the target substance. This invention, by real-time control of the rotational speed and direction of the erythrocyte microrotor, can achieve dynamic input, internal processing, and controllable output of different biological targets.

[0061] The present invention also provides a live animal intravascular biological microrouter constructed by the method described above.

[0062] This invention also provides a method for regulating the transmission path of target objects within the blood vessels of live animals based on the above-described scheme, comprising the following steps:

[0063] The method includes regulating the input of the target analyte into the biological microrouter and / or regulating the output of the target analyte from the biological microrouter;

[0064] The regulation of target input to the biological micro-router includes: with all ports closed, adjusting the direction of the circular trajectory scanning laser focus and reversing the rotation direction of the cell micro-rotor adjacent upstream of the port to make the port an input state, and the target is input into the biological micro-router from this port;

[0065] The output of the target substance from the biological micro-router includes: with all ports closed, adjusting the direction of the circular trajectory scanning laser focus and reversing the rotation direction of the adjacent cell micro-rotors downstream of the port to make the port in an output state, and the target substance is output from the biological micro-router.

[0066] The method described is not for therapeutic purposes.

[0067] In this invention, when the number of the biological microrouters is one, the distance between the biological microrouter and the target site of the target is preferably ≤70μm. This distance allows the target to be input into the microrouter and output to the target site. The target preferably includes one or more of cell nuclei, platelets, leukocytes, and nanomedicines; the cell nucleus is preferably the nucleus of a erythrocyte. This invention utilizes the biological microrouter to guide platelets to controllably stop hemostasis and to deliver leukocytes for targeted clearance or active delivery of antithrombotic nanomedicines. The method of this invention does not require direct laser irradiation of the target, and therefore is not strictly limited by the inherent material properties of the target.

[0068] In this invention, after the target object is input into the biological micro-router, it is preferred to further include: adjusting the rotation direction of the cell micro-rotor adjacent upstream of the port so that the port is in a closed state; after the target object is output from the biological micro-router, it is preferred to further include: adjusting the rotation direction of the cell micro-rotor adjacent downstream of the port so that the port is in a closed state.

[0069] In this invention, the method preferably further includes adjusting the rotation radius of the target object within the biological microrouter; the method for adjusting the rotation radius of the target object within the biological microrouter preferably includes: by adjusting the direction of the focal point of the circular trajectory scanning laser, reversing the rotation direction of any one cell microrotor, and setting one port to an output state, causing the rotating target object to undergo linear transport to the outside of the biological microrouter; before the target object is output from the biological microrouter, adjusting the port controlled by the cell microrotor to a closed state, so that the target object rotates within the biological microrouter with a larger radius; or, by moving the target object towards the center of the biological microrouter, the target object rotates within the biological microrouter with a smaller radius.

[0070] In this invention, after the target object is input into the biological micro-router, the biological micro-router exhibits powerful internal processing capabilities, allowing the target object to undergo controlled rotation, linear transmission, or be designed to separate. In this invention, when there are multiple or various target objects, these objects can be input or output sequentially from specific ports according to a preset path.

[0071] In one embodiment of the present invention, the bio-microrouter contains two non-overlapping target objects in the vertical direction, and the two target objects are closely located at the center of the microrouter and rotate together. The method further includes increasing the distance between the two target objects in the bio-microrouter, including: reversing the rotation direction of two spaced cell micro-rotors, setting the ports controlled by the two cell micro-rotors to an output state, and linearly transporting the two target objects towards the outside of the bio-microrouter towards the two output ports, thereby increasing the distance between the two target objects. This embodiment can create a breakpoint with zero flow velocity in the internal microflow field of the bio-microrouter; the breakpoint refers to the region where the flow field velocity inside the router is 0, and the target objects at the breakpoint are not affected by the flow field; when the two target objects are not located at the breakpoint but on both sides of the breakpoint, they will be affected by the flow field. Starting from the breakpoint, the two target objects can be transported in opposite directions.

[0072] In one embodiment of the present invention, the annular scanning optical potential well of any one cell microrotor is turned off, and the two cell microrotors upstream of the cell microrotor are rotated counterclockwise along the counterclockwise direction of the biological microrouter, while the two cell microrotors downstream of the cell microrotor are rotated clockwise. Then the target object is linearly transported toward the cell microrotor that is close to the one with the annular scanning optical potential well turned off.

[0073] In one embodiment of the present invention, the annular scanning optical potential well of any one cell microrotor is turned off, and the two cell microrotors upstream of the cell microrotor are rotated clockwise along the counterclockwise direction of the biological microrouter, while the two cell microrotors downstream of the cell microrotor are rotated counterclockwise. The target object is then linearly transported away from the cell microrotor whose annular scanning optical potential well is turned off.

[0074] In one embodiment of the present invention, by closing the annular scanning optical potential trap of any one cell microrotor and rotating the other four cell microrotors in a counterclockwise direction, a breakpoint with zero flow velocity can be created in the internal microflow field of the biological microrouter. The target object remains stationary at the breakpoint, while the target object not at the breakpoint is driven to rotate inside the biological microrouter.

[0075] In this invention, to avoid interference from other red blood cells to the biological micro-router, it is preferable to further include using a fixed optical potential well to capture other red blood cells outside the biological micro-router; or, using the other red blood cells as targets, the biological micro-router is used to change the transmission path of the other red blood cells.

[0076] The method of this invention utilizes endogenous red blood cells to construct a biocompatible microrouter within a living blood vessel. By real-time control of the rotation speed and direction of five red blood cells, dynamic input, internal processing, and controllable output of different targets within the blood vessel are achieved, thereby enabling dynamic routing selection for different targets. Furthermore, the biorouter of this invention achieves highly localized driving microfluidics without affecting surrounding cells, thus avoiding the indiscriminate delivery of all targets by large-scale flow fields.

[0077] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, provides a method for controlling the transmission path of a target within the blood vessels of a living animal based on scanning optical tweezers. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0078] Terminology Explanation:

[0079] Optical tweezers: Based on the photomomentum transfer caused by the interaction between a highly focused beam of light and matter, they generate photodynamic force on micro / nano particles and biological cells, thereby capturing them to the laser focus. By changing the spatiotemporal distribution of the focus, precise arrangement and rotational manipulation of multiple micro / nano particles and biological cells can be achieved.

[0080] Optical forces can be specifically divided into optical gradient forces and light scattering forces. Light scattering forces, along the direction of light propagation and proportional to the laser intensity, push micro / nano particles away along the optical axis. Optical gradient forces, pointing towards the laser focal point and proportional to the laser intensity gradient, can be further subdivided into optical gradient forces perpendicular to the optical axis and optical gradient forces along the optical axis. When micro / nano particles are near the focal point, they are first pulled towards the optical axis by the optical gradient force perpendicular to the optical axis, and then subjected to competing axial optical gradient forces and light scattering forces along the optical axis. When the axial optical gradient force dominates, stable capture and manipulation of micro / nano particles and biological cells can be achieved.

[0081] Optical flow manipulation: Optical fluid control studies how to control light and fluid at the micro-nano scale and use their interaction to regulate the parameters of the light field. At the same time, it can stably capture and precisely manipulate micro-nano particles and biological cells in microfluidics.

[0082] Scanning optical tweezers: Utilizing the interaction between an acousto-optic polarizer and a laser, rapid scanning of the laser focus across multiple positions on the focal plane can be achieved (maximum scanning frequency: 100 kHz). This allows for the creation of multiple optical potential wells using a single laser beam through time-division multiplexing, enabling the stable capture and precise alignment of multiple particles. By setting the laser focus to scan along a circular trajectory, the captured particles can rotate synchronously, with the rotation direction and speed dynamically adjustable.

[0083] Biophotonics: Based on light or other forms of radiation energy, it aims to understand the intrinsic working mechanisms of cells in living organisms, enabling the observation, detection, manipulation, and analysis of living tissues and biological cells, and further applying these to the diagnosis and treatment of clinical diseases.

[0084] Example 1

[0085] 1. Principle

[0086] The principle is as follows Figure 1 As shown, under the influence of optical gradient force, five red blood cells are captured one by one and then arranged into an equilateral pentagon. A circular scanning optical potential well is applied to each red blood cell. By scanning the laser focus along the circular trajectory, these red blood cells will dynamically rotate around their own axis under the influence of optical torque, and their rotation speed and direction can be controlled in real time. Each rotating red blood cell can be regarded as an endogenous cellular microrotor. Driven by their rotation, the surrounding blood will flow synchronously, thereby generating a local microfluidic field. By adjusting the rotation mode of the cellular microrotors, real-time and precise control of the direction and speed of the driving microfluidic flow at different locations can be achieved. Based on this approach, this embodiment will construct a biological microrouter inside a living blood vessel, which is expected to enable dynamic transport and routing of various targets within the body.

[0087] 2. Materials and Methods

[0088] 1) Apparatus

[0089] The device is constructed around the Tweez250si scanning optical tweezers system (SOT) (see patent CN111617390A for details on the device for regulating erythrocytes in the blood vessels of living animals), and is combined with an inverted optical microscope for real-time observation and recording of the experimental process. Figure 2The laser beam is selected as a continuous Gaussian beam with a wavelength of 1064 nm. First, the emitted laser beam interacts with an acousto-optic deflector (AOD, maximum scan rate 100 kHz) to achieve real-time, programmable adjustment of the spatiotemporal distribution of the laser focus on the focal plane. Then, the laser beam is broadened by a beam expander so that its diameter completely covers the entrance pupil of the inverted objective lens. After beam broadening, the laser beam is reflected upwards by a beam splitter and then refocused onto red blood cells in the blood vessels of the zebrafish's tail using a 60× immersion microscope objective (CFIApo, NA = 1.0), enabling multifunctional manipulation of the red blood cells.

[0090] Meanwhile, this embodiment uses a halogen white light source (D-LH) (12V, 100W) to emit illumination light from above, which is focused by a condenser and then irradiates the sample surface. The specific operation process is recorded by a high-speed charge-coupled device (CCD) camera and simultaneously displayed on a computer screen for real-time monitoring, image acquisition, and video recording.

[0091] In addition to capturing and manipulating individual red blood cells, the acousto-optic deflector can be programmed in real time via a software interface, allowing a laser beam to illuminate different locations at different times and repeatedly scan between those locations. Since the acousto-optic deflector's maximum scanning frequency can reach 100,000 Hz, it is possible to simultaneously manipulate multiple red blood cells.

[0092] To achieve red blood cell rotation, MATLAB software was used to merge the position coordinates of multiple optical potential wells in a ring-shaped scanning optical potential well into a patterned scanning sequence. Specifically, 3000 optical potential wells were arranged and merged into a circular scanning sequence with a diameter of 5.8 μm, forming a ring-shaped scanning optical potential well. The center position of the scanning trajectory corresponding to the scanning sequence can be moved in real time with the mouse as needed. When this scanning sequence is placed on a red blood cell that has been captured by a single optical potential well, the red blood cell will rotate because the laser will dynamically scan along the order set in the scanning sequence. By setting the optical potential wells in the sequence in the opposite order, the red blood cell will rotate in the opposite direction. Furthermore, by placing five circular optical potential wells on five red blood cells that have been captured and arranged in a pentagon, the assembly of a biological micro-router was achieved.

[0093] Figure 2In the image, the light source and condenser provide the illumination light required for observing the sample under bright field conditions. Beam splitters are dichroic mirrors that achieve spectral dispersion based on the wavelength of light transmitted or reflected. Long-pass dichroic mirrors highly reflect light below the cutoff wavelength while highly transmitting light above the cutoff wavelength; short-pass dichroic mirrors are the opposite, highly transmitting light below the cutoff wavelength while highly reflecting light above the cutoff wavelength. This embodiment uses a short-pass dichroic mirror, allowing illumination light with wavelengths below 800nm ​​to pass through while reflecting near-infrared laser light above 800nm. Acousto-optic deflectors generate mechanical waves in the crystalline medium, causing periodic changes in refractive index and creating a phase-type diffraction grating. If a laser beam is incident on the medium, the laser beam diffracts, and the intensity and direction of the diffracted light change with the intensity and wavelength of the mechanical wave. This principle allows the focal position and intensity of a 1064nm laser to be altered. Beam widening device: Composed of two convex lenses, the distance between them being the sum of their focal lengths, used to enlarge the laser's output diameter so that it completely covers the entrance pupil of the inverted objective lens. CCD camera: Short for charge-coupled device, used to acquire images in real time; the specific acquisition frequency can be set via a PC.

[0094] 2) Care and treatment of zebrafish

[0095] Adult zebrafish (90 days old) were purchased from Nanjing Zelinka Biotechnology Co., Ltd. (Nanjing, China). Following standard procedures, the zebrafish were fed with live marinated shrimp and cultured in clean tanks under a 28.5°C light / dark cycle for 14 hours.

[0096] 3) Preparation of nanomedicines

[0097] Mesoporous silica nanoparticle solution and urokinase were purchased from Huizhi Biotechnology Co., Ltd. (Shanghai, China). First, dried doxorubicin (5 mg) was added to the mesoporous silica nanoparticle solution (0.1 mg / mL, 5 mL), followed by sonication to obtain a homogeneous solution. Then, the solution was stirred in the dark for 24 h (100 rpm). Finally, the doxorubicin-loaded mesoporous silica nanoparticles (i.e., nanomedicine) were centrifuged and washed multiple times with PBS solution.

[0098] 4) Injecting nanomedicines into zebrafish

[0099] Zebrafish were general anesthetized for 8 minutes in a culture dish containing tricaine solution (200 mg / mL). Afterward, the zebrafish were transferred to 15 × 50 mm coverslips and fixed onto agarose slides. Nanomedicine was then administered using a solution with a concentration of 8 × 10⁻⁶ mg / mL. 6Dilute with phosphate buffer at a concentration of # / mL. After sonication (4800 rpm, 10 min), the monodisperse solution is loaded into a glass micropipette (outer diameter: 1.14 mm; inner diameter: 0.5 mm). The micropipette tip diameter is then stretched to 0.5–5 μm to ensure precise injection with negligible physiological damage. Finally, using a programmable nanoliter syringe (Nanojet III, Drummond, Inc.), 10 nL of the nanoparticle solution is injected into the posterior basal vein of the zebrafish.

[0100] 3. Operation

[0101] 1) Flexible assembly of biological microrouters in vivo

[0102] First, this embodiment investigated the assembly of a bio-microrouter in the blood vessels of a living zebrafish. This embodiment characterized the rotational flexibility of light-capturing erythrocytes. For example... Figure 3 As shown in Figure a, a red blood cell is captured and fixed in a blood vessel using an optical potential trap. Simultaneously, a set of laser beam focusing points was designed to introduce a circular scanning mode for the laser beam (red dashed circle). In this mode, the red blood cell begins to rotate clockwise with a rotation period of 0.9 s. At t = 1.2 s, the scanning direction is reversed, and the red blood cell begins to rotate counterclockwise with the same rotation period. This controlled rotation exhibits high stability, with an average rotation radius of 5.8 μm and an average speed of 7.0 rad / s. Figure 3 (b1 and b2 in the text). Furthermore, by controlling the scanning speed of the laser beam, the rotation speed (1.0–8.0 rad / s) can be adjusted in real time. Figure 3 (b3 in the middle).

[0103] Subsequently, this embodiment investigated the simultaneous rotation of multiple red blood cells. For example... Figure 3 As shown in Figure c, two cell microrotors are captured by placing two fixed optical potential traps and driven by a circular scanning laser beam as described above. Ultimately, they achieve synchronous clockwise rotation with a rotation period of 0.8 s. Similarly, three or four red blood cells can also be arranged into a designed pattern and rotate simultaneously. Figure 3 (d and e in the text). Finally, five red blood cells were arranged into an equilateral pentagon in the blood vessel to assemble a biological microrouter, in which the cell microrotors rotated clockwise for a period of 1.2 s ( Figure 3 (f1 to f3 in the diagram). Importantly, by adjusting the scanning direction and speed of the laser beam on the red blood cells, it is possible to control each red blood cell (e.g., red blood cell number 1) to rotate in the opposite direction or at a different speed, while other red blood cells remain unchanged. Figure 3 (f4 and f5 in the text). Therefore, independent control of individual biological components in a biological microrouter can be achieved to alter the microflow field and realize multifunctional routing in a precise and programmable manner.

[0104] 2) Performance characterization of biological microrouters

[0105] After the biological micro-router was assembled, its dynamic input, internal processing, and controllable output functions were characterized. Figure 4 Figures a and c in the diagram are schematics of the assembled biological micro-router, which can be viewed as a multipath router with five input and output ports. According to simulation results, the biological micro-router assembled in this embodiment can achieve the input or output of a target object from a specific port by changing the rotation direction of the corresponding cell micro-rotor. Figure 4 (a2 and c2 in the original text). To verify this hypothesis, a cell nucleus (extracted from a red blood cell, radius: 1 μm) was captured as a biological target and placed outside port 3 of the assembled biological micro-router. Figure 4 (b1 in the text). At t=2s, the microrotor of cell 3 was switched to rotate counterclockwise ( Figure 4 (b2 in the text). Finally, the target object was successfully input into the biological micro-router via port 3. Figure 4 (b3 in the text). Furthermore, by switching the rotation direction of the cell microrotor to counterclockwise, the target object can be output from port 5 (b3 in the text). Figure 4 (d) Furthermore, the five targets can be programmably input or output one by one from their respective ports. These results demonstrate that the bio-microrouters assembled in blood vessels can effectively realize the designed target input and output functions.

[0106] After the target object is input, the assembled bio-micro-router exhibits powerful internal processing capabilities. Under these capabilities, the target object can undergo controlled rotation, linear transmission, and designed separation, much like an intelligent macro-router capable of performing complex signal processing. Figure 4 (e). By rotating all the cell microrotors in the same direction, the input target begins to rotate inside the biological microrouter, and the rotation direction of the target can be switched by changing the rotation direction of the cell microrotors. Figure 4 f in the middle.

[0107] Furthermore, the target object's rotation radius can be adjusted in real time. For example... Figure 4 As shown in g, at t = 5.4 s, by switching the rotation direction of cell microrotor #2, the rotating target object is linearly transported towards the outside of the biological microrouter. After linear transport for a certain distance, before the target object exits the biological microrouter, the port controlled by the cell microrotor is adjusted to a closed state. Then, the target object is rotated again with a larger radius, i.e., the radius increases from 0 to 6 μm. Similarly, by moving the target object towards the center of the biological microrouter, the rotation radius of the target object can be controlled to decrease, for example, from 5 μm to 0 μm. Figure 4 (h in the example). Specifically: at 0s, the target object is located at the inner edge of the biological micro-router and rotates along the inner edge of the router with a radius of 5. When the target object moves to the vicinity of port 5 at 12.8s, this embodiment changes the rotation direction of cell micro-rotor No. 5 to counterclockwise, so that a flow field is generated at port 5 towards the inner center of the biological micro-router, thereby causing the target object to move towards the inner center of the biological micro-router and reach the inner center at 17.6s. After that, this embodiment changes the rotation direction of cell micro-rotor No. 5 back to clockwise. At this time, the target object will rotate with a radius of 0 at the inner center of the biological micro-router.

[0108] The assembled biological microrouters can also be used to process two targets simultaneously. For example... Figure 4 As shown in diagram i, the two targets are closely located at the center of the bio-microrouter and rotate together. Using a similar method to the above, increasing the rotation radius to 7 μm clearly shows the separation of the two targets. As shown in diagram k3, the two targets initially do not overlap near the breakpoint. By setting cell microrotors 1 and 3 to rotate counterclockwise and cell microrotors 2, 4, and 5 to rotate clockwise, a flow field is generated at ports 1 and 3 outward along the bio-microrouter, causing the two targets to separate and move towards ports 1 and 3. If they overlap vertically, the bio-microrouter cannot separate them temporarily because the scanning optical tweezers system used primarily manipulates objects in a two-dimensional plane. Therefore, the bio-microrouter constructed in this embodiment mainly performs routing selection for targets in a two-dimensional plane.

[0109] Furthermore, by rotating red blood cells 2 and 3 clockwise, and red blood cells 1 and 5 counterclockwise, while keeping red blood cell 4 stationary, the two target objects can be transported together to the right. Figure 4 In k1), by changing the rotation direction of the four cell microrotors, their transport direction can be changed to leftward transport. Figure 4 (k2 in the middle).

[0110] When red blood cells 2, 4, and 5 are rotated clockwise, and red blood cells 1 and 3 are rotated counterclockwise, a breakpoint with zero flow velocity is created in the internal micro-flow field of the biological micro-router (i.e., the breakpoint refers to the region where the flow velocity inside the router is 0, and the target object at the breakpoint is not affected by the flow field). When two target objects are not located at the breakpoint but on either side of it, they will be affected by the flow field. Starting from the breakpoint, the two target objects can be transported in opposite directions. Figure 4 (k3 in the middle).

[0111] Furthermore, by keeping red blood cell #1 stationary and rotating the other four red blood cells counterclockwise, target object #1 can remain stationary at the breakpoint, while target object #2 is driven to rotate within the biological micro-router. Figure 4(i) These results demonstrate that the assembled bio-microrouter possesses dynamic internal transport and separation capabilities due to the programmable control of the five cellular microrotors.

[0112] Based on its programmable internal processing capabilities, the biological micro-router was further subjected to various target studies.

[0113] The test demonstrates diverse input / output capabilities. First, it shows the scenario where the target object is input from a specific port and outputs from different ports. Figure 5 a) in the example. Figure 5 As shown in a2-a4, by rotating red blood cell number 2 counterclockwise, the target object (cell nucleus) near port 2 can be input into the biological micro-router. Then, at t=5.3s, the rotation direction of red blood cell number 2 is changed back to clockwise. Figure 5 (a4 in the text). The target object begins to rotate at the center of the biological micro-router ( Figure 5 The red blood cell (a5) was transported to port 1 at t = 7.6 s. At this time, the rotation direction of red blood cell #2 was changed to counterclockwise, resulting in the successful output of the target material (a5) from port 1. Figure 5 (a6 in the text). Similarly, the target object input from the same port (i.e., port 2) can also be transported to ports 3, 4, or 5 within the biological micro-router via linear transport and rotation around the center. Then, by switching the rotation direction of red blood cells 4, 5, or 1, the target object is output from the corresponding port. Figure 5 (a7~a9 in the text).

[0114] Furthermore, multiple targets can be input from different ports, and multiple targets can be output from the same port. For example... Figure 5 As shown in b, targets 1, 2, and 3 are input from ports 5, 1, and 2, respectively. Inside the biological micro-router, the three targets are transported to port 4 via linear transport, and then output from that port by changing the rotation direction of red blood cell 5.

[0115] Furthermore, multiple targets can be input and output through the same transmission path. For example... Figure 5 As shown in Figure c, by continuously switching the rotation direction of red blood cell 2, three target objects were input from port 2. These three target objects underwent linear transport within the biological micro-router. By changing the rotation direction of red blood cell 5, these three target objects could be output from port 4 along similar trajectories. In addition, target objects can also be input and output through different transmission paths. Figure 5 (d) In this context, target 1 is input through port 1, undergoes linear transmission within the bio-micro router, and is finally output through port 3. Target 2, on the other hand, can be input through port 5 into the bio-micro router and output from the nearby port 4.

[0116] Besides the cell nucleus, larger blood cells, including platelets and white blood cells, also possess this ability to be routed selectively. For example... Figure 5 As shown in Figure e, by rotating red blood cells 2 and 5 counterclockwise and red blood cells 1, 3, and 4 clockwise, a platelet can be input from port 2 and output from port 4. The platelet's transport distance is 45 μm within 3 seconds. Using the same method, a white blood cell with a diameter of 8.5 μm can also be transported along a similar route. Figure 5 (f) Furthermore, platelets and cell nuclei can be dynamically routed to either branch, thus enabling controlled separation of various targets. Therefore, the assembled biological micro-router possesses flexible routing capabilities for various biological targets.

[0117] 3) Biomedical applications of bio-microrouters

[0118] Based on the above functions, the assembled bio-microrouter can be used in some biomedical applications, such as guiding platelets to controllable hemostasis, delivering leukocytes for targeted clearance, and actively delivering antithrombotic nanomedicines. Figure 6 a) in the example. Figure 6 As shown in b, four platelets move with the blood flow, and there is a damaged area (laser-burned) on the blood vessel wall. To guide these platelets to the damaged blood vessel wall, a bio-microrouter was assembled in situ using endogenous red blood cells. Port 1 of the bio-microrouter was designed near the platelets, while port 3 faced the damaged area. By rotating red blood cells 2 and 3 clockwise and red blood cells 1, 4, and 5 counterclockwise, these platelets were sequentially input from port 2 and output through port 3 to reach the damaged blood vessel wall.

[0119] Considering the ability of biological microrouters to separate target objects, this embodiment will utilize it to transmit different target objects to perform different biological tasks. For example... Figure 6 As shown in c1, platelets and leukocytes are located at the center of the blood vessel, with damaged areas and cell debris on the left and right sides, respectively. A biological micro-router is assembled and programmed to receive these two targets through port 2 and output platelets and leukocytes through ports 1 and 4, respectively. In this way, platelets are guided to the damaged blood vessel for hemostasis. Figure 6 (c2) while leukocytes are guided to cell debris and targeted for clearance through phagocytosis. Figure 6 (c3 in the middle).

[0120] Since bio-microrouters can induce microfluidics to drive the movement of various targets in specific directions, this embodiment further explores their potential for active delivery of nanomedicines. Mesoporous silica nanoparticles (average diameter 600 nm) loaded with urokinase were used as antithrombotic nanomedicines and injected into blood vessels via microinjection. At t=0s, there was a flow of nanomedicine within the main blood vessel, with a branch on its left side (…). Figure 6 (d1 in the example). In this embodiment, a bio-microrouter was then assembled in front of the nanomedicine. By changing the rotation direction of red blood cells 1 and 5, the nanomedicine was successfully delivered to the branch at t = 13.4 s. Figure 6 (d2 in the original text). Since the movement of nanomedicines is driven by microfluidics, the number of nanomedicines that can be transported via a bio-microrouter should not be limited to one. To test this hypothesis, a bio-microrouter was assembled between a thrombus and a group of nanomedicines. Figure 6 (e1 in the text). After changing the rotation direction of red blood cells 2 and 3, the nanomedicine is input through port 1, output through port 3, and delivered to the thrombus on the blood vessel wall, thus achieving large-scale targeted delivery of the nanomedicine. Figure 6 (e3 in the middle).

[0121] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for constructing intravascular biological microrouters in living animals, characterized in that, Includes the following steps: Red blood cells are captured and arranged into biological microrouters using an optical potential trap; the shape of the biological microrouters is an equilateral pentagon; each biological microrouter consists of 5 red blood cells; Each red blood cell, as a cellular microrotor, is located at the vertex of an equilateral pentagon; the gaps between adjacent cellular microrotors in each of the biological microrouters constitute ports; A ring-shaped scanning optical potential trap is applied to each of the cell microrotors; When the laser focus is scanned along the circular trajectory, the cell microrotor rotates; the direction of the circular trajectory scanning laser focus is the same as the direction of the cell microrotor's rotation. In the biological micro-router, in a counterclockwise direction, the cell micro-rotor adjacent upstream of the port is used to control the port to be in an input state or a closed state, and the cell micro-rotor adjacent downstream of the port is used to control the port to be in an output state or a closed state. With all ports closed, the rotation direction of each cell micro-rotor in the biological micro-router is either clockwise or counterclockwise. Each red blood cell in the biological micro-router is independently controlled; the control method for a single red blood cell is: by adjusting the scanning direction and speed of the laser beam on the red blood cell, one red blood cell is controlled to rotate in the opposite direction or at a different speed, while the other red blood cells remain unchanged.

2. The method according to claim 1, characterized in that, The rotation speed of the cell microrotor is 1~18 rad / s.

3. The method according to claim 1, characterized in that, The optical potential well that captures red blood cells is a fixed optical potential well; the fixed optical potential well captures the center position of the red blood cells; the annular scanning optical potential well is centered on the center of the red blood cells; the diameter of the annular scanning optical potential well is less than or equal to the diameter of the red blood cells.

4. The method according to claim 1, characterized in that, Each of the aforementioned annular scanning optical potential wells comprises 3000 optical potential wells.

5. The method according to claim 1, characterized in that, The scanning frequency of the annular scanning optical potential well is ≤100000Hz.

6. The method according to claim 1, characterized in that, The side length of the biological microrouter is the distance between the center points of adjacent cell microrotors; the ratio of the biological microrouter to the blood vessel diameter is ≤0.

75.

7. The intravascular biological microrouter for living animals constructed by the method described in any one of claims 1 to 6.

8. A method for regulating the transmission path of a target within the blood vessels of a living animal based on the intravascular biological microrouter described in claim 7, characterized in that, Includes the following steps: The method includes regulating the input of the target analyte into the biological microrouter and / or regulating the output of the target analyte from the biological microrouter; The regulation of target input to the biological micro-router includes: with all ports closed, adjusting the direction of the circular trajectory scanning laser focus and reversing the rotation direction of the cell micro-rotor adjacent upstream of the port to make the port an input state, and the target is input into the biological micro-router from this port; The output of the target substance from the biological micro-router includes: with all ports closed, adjusting the direction of the circular trajectory scanning laser focus and reversing the rotation direction of the adjacent cell micro-rotors downstream of the port to make the port in an output state, and the target substance is output from the biological micro-router. The method described is not for therapeutic purposes.

9. The method according to claim 8, characterized in that, After the target object is input into the biological micro-router, the method further includes: adjusting the rotation direction of the cell micro-rotor adjacent to the upstream of the port so that the port is in a closed state; After the target object is output to the biological micro-router, the method further includes: adjusting the rotation direction of the adjacent cell micro-rotor downstream of the port so that the port is in a closed state.

10. The method according to claim 8, characterized in that, The method also includes adjusting the rotation radius of the target object within the biological micro-router; The method for controlling the rotation radius of the target object within the bio-microrouter includes: adjusting the direction of the circular trajectory scanning laser focus, reversing the rotation direction of any one cell microrotor, and setting one port to an output state, causing the rotating target object to undergo linear transport to the outside of the bio-microrouter; before the target object is output from the bio-microrouter, adjusting the port controlled by the cell microrotor to a closed state, causing the target object to rotate within the bio-microrouter with a larger radius; or, by moving the target object towards the center of the bio-microrouter, causing the target object to rotate within the bio-microrouter with a smaller radius.

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