In vivo capture system for cells to be tested and working method thereof

Abstract

The present invention discloses an in vivo capture system for cells to be tested and a working method thereof, the system comprising an immune micro-nano bubble supply device, an acoustic resonance immune guide body in vivo delivery device, an ultrasonic emitting device and an acoustic resonance immune guide body; the immune micro-nano bubble supply device provides immune micro-nano bubbles coupled with antibodies, the immune micro-nano bubbles are injected into the body to specifically bind to the cells to be tested to form a bubble complex; the guide body in vivo delivery device delivers the acoustic resonance immune guide body to the in vivo area to be tested; the ultrasonic emitting device emits ultrasonic waves to stimulate the acoustic resonance immune guide body in the body to generate a local strong field; the surface coupling of the acoustic resonance immune guide body can identify antibodies of the cells to be tested, and when being delivered to the body, it specifically binds to the cells to be tested to capture the cells to be tested, and uses the local strong field to adsorb the bubble complex to capture the cells to be tested. The present invention can increase the number of captured trace cells to be tested in the body and improve the accuracy of subsequent detection.

Description

Technical Field

[0001] The invention belongs to the technical field of in vivo cell detection, and in particular relates to an in vivo capture system for cells to be detected and a working method thereof. Background Art

[0002] In recent years, despite the progress made in cancer treatment, tumor metastasis remains the ultimate challenge in the fight against cancer treatment and the main culprit for cancer-related deaths. Circulating tumor cells (CTCs) are important mediators of tumor metastasis. During the development of tumors, CTCs are released into the blood circulation by the primary tumor or metastatic lesions, and then colonize in distant tissues or organs of the body to form new metastatic lesions. As a safe and reliable biological marker, CTCs have the advantages of being non-invasive, easy to obtain, and highly reproducible. Their application in the field of malignant tumors has developed rapidly, and related research covers many fields such as early diagnosis, treatment, prognosis evaluation, and pathogenesis of tumors. However, although the blood of cancer patients may contain hundreds or thousands of CTCs, the traditional detection method of 5-10 ml of blood samples (accounting for only 0.1% of the total blood volume) usually contains only a small amount of CTCs. This order of magnitude of tumor cells is not enough to conduct a comprehensive detection of biological behaviors such as tumor molecular biological heterogeneity and drug-resistant mutations. Low numbers of CTCs may lead to false-negative results in tumor blood samples, especially in early tumor detection or tumor recurrence detection, thus leading to misguided clinical decision-making and causing irreparable losses in the treatment and survival prognosis of cancer patients.

[0003] At present, based on the physical and biological characteristics of CTCs, CTC detection technology can be divided into three categories, namely: CTC detection method based on nucleic acid expression, CTC detection method based on cell physical properties, and CTC detection method based on cell surface antibodies.

[0004] The detection of CTC by nucleic acid expression is mainly based on the fact that CTC has DNA or RNA expression that is different from blood component cells, thereby detecting free nucleic acids in the serum of tumor patients. However, the nucleic acids carried by tumor apoptotic cells, exosomes, and other nucleated cells in peripheral blood can significantly affect the detection results, resulting in obvious disadvantages of this technology. It is currently believed that compared with blood component cells, CTC has a larger volume and density, and the charge distribution on the cell surface is also different from other cells. In addition, some CTCs may also show different characteristics such as cell activity. These specific differences have gradually been recognized and applied to cell detection and screening. However, it is difficult to accurately detect CTC using this detection method because it is easily interfered by various internal and external factors. With the large-scale development of related research on tumor cell membrane proteins and related functional verification, a theoretical basis and new options have been provided for the detection and sorting of CTCs, thereby significantly improving the sensitivity and specificity of CTC detection technology based on cell surface antibodies. The most widely used technology currently relies mainly on CTC cell surface specific antibodies.

[0005] Epithelial cell adhesion molecule (EpCAM) is widely used in CTC detection based on cell surface antibodies because it is expressed in almost all epithelial cells but not in blood cells. Epithelial tumor cells are enriched and bound by immunomagnetic beads coupled with specific anti-EpCAM antibodies, and then screened by a specific magnetic field. They have been used to detect CTCs in malignant tumors such as lung cancer, breast cancer, and colorectal cancer. At present, there are two representative CTC detection technologies developed based on this principle, namely the CellSearch system, a commercial product produced by Veridex approved by the U.S. Food and Drug Administration (FDA) and the China Food and Drug Administration (CFDA), and the CellCollector sampling needle produced by Gilupi, Germany, the only one in the world that captures CTCs in vivo and approved by CFDA.

[0006] Although the CellSearch system is a relatively standard CTC detection method among existing detection technologies and has begun to be applied to some clinical trials, the main disadvantage of this system is its low sensitivity (approximately only 1 CTC cell is detected per milliliter of blood), and a single test usually requires a large blood sample, and effective detection of CTCs can only be achieved in the peripheral blood of patients with distant tumor metastasis. See: Andree, KC; van Dalum, G.; Terstappen, LW Challenges incirculating tumor cell detection by the CellSearch system. Mol Oncol 2016; 10: 395-407.

[0007] Compared with the CellSearch system, the CellCollector sampling needle has been shown to have advantages in terms of cell quantity acquisition and sensitivity in CTC detection. However, the CellCollector sampling needle has limited ability to capture CTC in vivo, and its ability to attract and capture CTC cells flowing in blood vessels is poor. It can only capture CTC cells flowing through the surface of the sampling needle, but cannot capture CTC cells far away from the sampling needle. See: Vermesh, O.; Aalipour, A.; Ge, TJ; Saenz, Y.; Guo, Y.; Alam, IS; Park, SM; Adelson, CN; Mitsutake, Y.; Vilches-Moure, J., et al. An intravascular magnetic wire for the high-throughput retrieval of circulating tumor cells in vivo. Nat Biomed Eng 2018; 2: 696-705. Summary of the invention

[0008] In view of the shortcomings of the prior art, the present invention provides an in vivo capture system for cells to be tested and a working method thereof, so as to increase the number of captured trace cells to be tested in vivo (such as circulating tumor cells CTC) and improve the accuracy of subsequent detection.

[0009] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions:

[0010] A system for capturing cells in vivo, the system comprising an immune micro-nano bubble supply device, an acoustic resonance immune guide body in vivo delivery device, an ultrasonic emission device and an acoustic resonance immune guide body; wherein,

[0011] The immune micro-nano bubble supply device is used to provide immune micro-nano bubbles, the surface of which is coupled with antibodies that can identify cells to be tested, and the immune micro-nano bubbles are used to be injected into the area to be tested in the body and specifically bind to the cells to be tested to form immune micro-nano bubble complexes;

[0012] The acoustic resonance immune guide body in vivo delivery device is used to deliver the acoustic resonance immune guide body from outside the body to the area to be tested inside the body and from the area to be tested inside the body to outside the body;

[0013] The ultrasonic emitting device is used to emit ultrasonic waves outside the body to stimulate the acoustic resonance immune guide located inside the body to generate a local strong field;

[0014] The surface of the acoustic resonance immune guide is coupled with antibodies that can recognize cells to be tested, which are used to specifically bind to cells to be tested and directly capture cells to be tested when being transported to the area to be tested in the body, and use the local strong field to adsorb the immune micro-nano bubble complex to capture cells to be tested.

[0015] Among them, the immune micro-nano bubble supply device is a microfluidic cavity device, including a primary cavity, a secondary cavity and a tertiary cavity connected in sequence; the primary cavity includes a gas cavity, a liquid lipid cavity, a bubble-forming cavity and an antibody delivery cavity; the secondary cavity is a cavity for coupling and assembling antibodies and micro-nano bubbles; the tertiary cavity is a cavity for screening and obtaining immune micro-nano bubbles with antibodies coupled on the surface that can recognize the cells to be tested.

[0016] Wherein, the diameter of the immune micro-nano bubbles is 1 μm to 10 μm.

[0017] The material of the acoustic resonance immune guide is a flexible material; the shear wave velocity of the acoustic resonance immune guide is smaller than the longitudinal wave velocity of the body fluid in the area to be tested in the body.

[0018] Wherein, the material of the acoustic resonance immune guide is a biomedical polymer material.

[0019] Wherein, the acoustic resonance immune guide body is a hollow or solid linear guide wire; the cross section of the acoustic resonance immune guide body is a triangle, a circle or a rectangle.

[0020] The surface of the acoustic resonance immune guide is covered with a hydrophilic or hydrophobic coating, and antibodies capable of identifying cells to be tested are coupled and assembled on the coating.

[0021] The circumferential diameter of the acoustic resonance immune guide is 50 μm to 500 μm, and the operating frequency for generating a local strong field mode is 1 MHz to 10 MHz.

[0022] Among them, the acoustic resonance immune guide in vivo delivery device is a puncture outer sheath, and the pre-installed puncture needle is used to puncture the in vivo test area through the puncture outer sheath, and the acoustic resonance immune guide is delivered to the internal test area after internal puncture through the puncture outer sheath.

[0023] The ultrasonic transmitting device comprises a signal generator, a power amplifier and an ultrasonic transducer. The signal generated by the signal generator is amplified by the power amplifier and then stimulates the ultrasonic transducer to transmit ultrasonic waves.

[0024] The in vivo capture system for cells to be tested further comprises a microfluidic cell sorting and detection device for detecting the cells to be tested captured by the acoustic resonance immune guide.

[0025] Wherein, the cells to be detected are circulating tumor cells, the antibodies capable of recognizing the cells to be detected are antibodies that recognize tumor-specific antigens, and the in vivo region to be detected is a blood vessel.

[0026] Another aspect of the present invention provides a working method of the in vivo capture system for cells to be detected as described above, comprising:

[0027] Controlling the immune micro-nano bubble supply device to prepare immune micro-nano bubbles with antibodies coupled on the surface that can recognize cells to be tested, and injecting the immune micro-nano bubbles into the area to be tested in vivo, so that the immune micro-nano bubbles and the cells to be tested specifically bind to form immune micro-nano bubble complexes;

[0028] Controlling the acoustic resonance immune guide body delivery device to deliver the acoustic resonance immune guide body with antibodies coupled to the surface capable of recognizing the cells to be tested to the area to be tested in the body, so that the acoustic resonance immune guide body specifically binds to the cells to be tested and directly captures the cells to be tested;

[0029] Controlling the ultrasonic emitting device to emit ultrasonic waves to stimulate the acoustic resonance immune guide to generate a local strong field, so that the acoustic resonance immune guide adsorbs the immune micro-nano bubble complex to capture the cells to be tested;

[0030] The acoustic resonance immune guide body in vivo delivery device is controlled to deliver the acoustic resonance immune guide body after capturing the cells to be tested to the outside of the body.

[0031] The in vivo capture system for cells to be tested provided in an embodiment of the present invention delivers immune micro-nano bubbles with antibodies capable of identifying cells to be tested coupled to their surfaces and an acoustic resonance immune guide into an in vivo region to be tested. On the one hand, the acoustic resonance immune guide can specifically bind to the cells to be tested to directly capture the cells to be tested. On the other hand, under the excitation of ultrasonic waves, the acoustic resonance immune guide is used as a "secondary sound source" to generate a local sound field to adsorb and enrich the immune micro-nano bubbles that are bound to the cells to be tested, thereby further capturing a larger number of cells to be tested, thereby increasing the number of trace cells to be tested (such as circulating tumor cells CTC) captured in the body and improving the accuracy of subsequent detection.

[0032] Taking the capture of CTC as an example, compared with the CellSearch system in the prior art, the present invention can continuously recruit and capture CTCs during the continuous circulation of blood in the body, thereby continuously increasing the local concentration of CTC cells and capturing more CTC cells. Compared with the CellSearch system in the prior art and the CellCollector sampling needle, the present invention can not only capture CTC cells flowing through the surface of the acoustic resonance immune guide, but also capture CTC cells within a certain distance around the resonance immune guide through the adsorption force of the local strong field, and can capture more CTC cells.

[0033] In addition, acoustic manipulation mainly utilizes the acoustic radiation force, drag or shear force caused by acoustic flow, etc. on particles in the acoustic field to achieve functions such as capture, arrangement, movement, rotation and release of particles. Since ultrasound has been widely used in clinical imaging detection, and it has been confirmed that low-intensity ultrasound does not cause significant damage to the body, ultrasonic manipulation of particles is a non-contact, non-invasive and convenient manipulation method. The capture of CTCs by acoustic manipulation of immune micro-nano bubbles does not cause cell damage and ensures the biological activity of CTCs. That is, the in vivo capture system for cells to be tested and its working method provided by the present invention are safer and more reliable. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 is a schematic structural diagram of an in vivo capture system for cells to be tested according to an embodiment of the present invention;

[0035] Figure 2 is a diagram showing that the immune micro-nano bubbles in an embodiment of the present invention are coupled with antibodies capable of recognizing cells to be tested;

[0036] Figure 3 is a diagram showing that the immune micro-nano bubbles and the cells to be tested are combined to form an immune micro-nano bubble complex in an embodiment of the present invention;

[0037] Figure 4 is a schematic structural diagram of an immune micro-nano bubble supply device in an embodiment of the present invention;

[0038] Figure 5 is an exemplary illustration of the structure of the acoustic resonance immune guide body delivery device and the process of delivering the acoustic resonance immune guide body in an embodiment of the present invention;

[0039] Figure 6 is a schematic structural diagram of an ultrasonic transmitting device in an embodiment of the present invention;

[0040] Figure 7 is a schematic diagram of the structure of an acoustic resonance immune guide in an embodiment of the present invention;

[0041] Figure 8 is a schematic structural diagram of a microfluidic cell sorting and detection device according to an embodiment of the present invention;

[0042] Fig. 9 is a work flow chart of the in vivo capture method of cells to be tested according to an embodiment of the present invention;

[0043] Figures 10 to 12 This is a diagram showing the force distribution of immune micro-nano bubbles of different diameters around an acoustic resonance guide with a diameter of 300 microns and a circular cross-section;

[0044] Fig.13 It is an enlarged photographic illustration of the capture of cells to be tested by the acoustic resonance guide in an embodiment of the present invention. DETAILED DESCRIPTION

[0045] In order to make the purpose, technical scheme and advantages of the present invention clearer, the specific embodiments of the present invention are described in detail below in conjunction with the accompanying drawings. Examples of these preferred embodiments are illustrated in the accompanying drawings. The embodiments of the present invention shown in the accompanying drawings and described according to the accompanying drawings are merely exemplary, and the present invention is not limited to these embodiments.

[0046] It should also be noted that, in order to avoid obscuring the present invention due to unnecessary details, only structures and / or processing steps closely related to the solutions according to the present invention are shown in the accompanying drawings, while other details that are not closely related to the present invention are omitted.

[0047] Figure 1 FIG. 1 is a schematic diagram of the structure of the in vivo capture system of cells to be tested according to an embodiment of the present invention. Figure 1 As shown, the in vivo capture system of cells to be tested includes: an immune micro-nano bubble supply device 10, an acoustic resonance immune guide in vivo delivery device 20, an ultrasonic emitting device 30 and an acoustic resonance guide 40. Preferably, the capture system of this embodiment also includes a microfluidic cell sorting detection device 50.

[0048] The immune micro-nano bubble supply device 10 is used to provide immune micro-nano bubbles. Figure 2 and Figure 3The surface of the immune micro-nano bubble 1 is coupled with an antibody 2 capable of recognizing a cell to be detected, and the immune micro-nano bubble 1 is used to be injected into a region to be detected in the body and specifically binds to a cell to be detected 3 to form an immune micro-nano bubble complex. In a specific technical solution, the cell to be detected 3 is, for example, a circulating tumor cell (CTC), the antibody 2 capable of recognizing a cell to be detected is an antibody that recognizes a tumor-specific antigen, and the region to be detected in the body is a blood vessel.

[0049] Specifically, the immune micro-nano bubble supply device 10 is a microfluidic cavity device, such as Figure 4 As shown, the microfluidic cavity device includes a primary cavity 11, a secondary cavity 12 and a tertiary cavity 13 which are connected in sequence.

[0050] See also Figure 4 The primary cavity 11 includes a gas cavity 11a, a liquid lipid cavity 11b, a bubbling cavity 11c and an antibody delivery cavity 11d. The gas cavity 11a is used to supply an inert gas G, such as perfluoropropane, to form the inner core of the immune micro-nano bubble; the liquid lipid cavity 11b is used to supply a liquid lipid material L, such as phospholipid, to form the outer shell of the immune micro-nano bubble; the bubbling cavity 11c is connected to the output ends of the gas cavity 11a and the liquid lipid cavity 11b, and the bubbling cavity 11c is a cavity where the inert gas G and the liquid lipid material L are combined to form the immune micro-nano bubble 1; the antibody delivery cavity 11d is used to supply antibodies 2 that can identify the cells to be tested. The secondary cavity 12 is connected to the output end of the bubble forming cavity 11c and the antibody delivery cavity 11d. The secondary cavity 12 is a cavity for coupling and assembling the antibody 2 and the immune micro-nano bubble 1. An ultrasonic device 14 is arranged outside the cavity at the end of the secondary cavity 12, and the immune micro-nano bubble assembled with the antibody is sorted by emitting ultrasonic waves 15. The tertiary cavity 13 is connected to the output end of the secondary cavity 12. The tertiary cavity 13 is a cavity for screening and obtaining immune micro-nano bubbles with antibodies coupled on the surface that can identify the cells to be tested. The tertiary cavity 13 is used to screen and remove immune micro-nano bubbles 1 with uniform particle size. Furthermore, the microfluidic cavity device also includes an excretion channel 16, which is used to discharge antibodies, micro-nano bubbles that have failed to be coupled and assembled, and immune micro-nano bubbles coupled with antibodies whose particle size does not meet the requirements.

[0051] Compared with the traditional technology for producing micro-nano bubbles, the microbubbles produced by the flow control cavity device of the present invention can achieve uniform and single particle size, thereby greatly optimizing the in vivo capture efficiency of the micro-nano bubbles.

[0052] The immune micro-nano bubbles provided in the embodiment of the present invention are lipid immune micro-nano bubbles, which include a lipid shell coupled with antibodies capable of recognizing cells to be detected and an inert gas core, and the diameter thereof is preferably in the range of 1 μm to 10 μm.

[0053] In a preferred embodiment, the immune micro-nano bubbles can also be chemically or biologically modified to increase their adhesion efficiency and targeting.

[0054] The acoustic resonance immune guide body delivery device 20 is used to deliver the acoustic resonance immune guide body 40 from outside the body to the area to be tested inside the body, or from the area to be tested inside the body to outside the body.

[0055] In this embodiment, refer to Figure 5 The acoustic resonance immune guide body in vivo delivery device 20 is specifically a puncture outer sheath tube 4, which is equipped with a puncture needle 5. The puncture outer sheath tube 4 includes a puncture needle port 4a, a guide body port 4b and a puncture port 4c. The process of the acoustic resonance immune guide body in vivo delivery device 20 delivering the acoustic resonance immune guide body 40 into the body is specifically as follows: first, the puncture needle 5 is placed into the puncture outer sheath tube 4 from the puncture needle port 4a, and the puncture port 4c is aligned with the target area (such as a blood vessel) for puncture. After the puncture is successful, the puncture needle 5 is withdrawn, and at this time, the acoustic resonance immune guide body 40 is placed into the puncture outer sheath tube 4 from the guide body port 4b, and is delivered to the punctured target area through the puncture port 4c.

[0056] The ultrasonic emitting device 30 is used to emit ultrasonic waves outside the body to stimulate the acoustic resonance immune guide 40 located inside the body to generate a local strong field.

[0057] In this embodiment, refer to Figure 6 The ultrasonic transmitting device 30 includes a signal generator 31, a power amplifier 32 and an ultrasonic transducer 33. The signal generated by the signal generator 31 is amplified by the power amplifier 32 to stimulate the ultrasonic transducer 33 to emit ultrasonic waves.

[0058] Specifically, the ultrasonic transducer 33 can be a single-element ultrasonic transducer, a phased array ultrasonic transducer, a linear array ultrasonic transducer, or a convex array ultrasonic transducer; wherein the resonance frequency of the acoustic resonance immune guide 40 determines the driving frequency of the ultrasonic emission, thereby determining the center frequency of the ultrasonic transducer 33. The signal generated by the signal generator 31 can be a continuous sinusoidal signal or a pulsed sinusoidal signal. In one embodiment, the signal generator 31 can be a programmable signal generator (AFG3021, Tectronix), and the power amplifier can be a 50dB linear power amplifier (325LA, ENI). The signal generator 31 generates a sinusoidal continuous signal, and the sinusoidal signal excites the ultrasonic transducer 33 to generate ultrasonic waves after passing through the power amplifier 32.

[0059] Among them, see Figure 7, the surface of the acoustic resonance immune guide 40 is coupled with antibodies 41 that can identify cells to be tested, and at least the part that is delivered to the target area is coupled with antibodies 41 that can identify cells to be tested; further, in this embodiment, the surface of the acoustic resonance immune guide 40 is first coated with a hydrophilic or hydrophobic coating 44, and the antibodies 41 that can identify cells to be tested are coupled and assembled on the coating 44. The acoustic resonance immune guide 40 is also provided with an insertion mark point 42 and an exit mark point 43. The acoustic resonance immune guide 40 is used to specifically bind to the cells to be tested and directly capture the cells to be tested when it is delivered to the area to be tested in the body, and the immune micro-nano bubble complex is adsorbed by the local strong field to capture the cells to be tested.

[0060] Combination Figure 5 and Figure 7 When the acoustic resonance immune guide 40 is placed into the puncture outer sheath 4 from the guide port 4b, the insertion mark point 42 of the acoustic resonance immune guide 40 is brought in to be flush with the guide port 4b. After the acoustic resonance immune guide 40 has completed capturing the cells to be tested, the acoustic resonance immune guide 40 is slowly withdrawn from the guide port 4b until the exit mark point 43 is flush with the guide port 4b, and then the entire puncture outer sheath 4 is withdrawn from the target area.

[0061] The material of the acoustic resonance immune guide is a flexible material and a biomedical polymer material, such as polylactic acid-glycolic acid copolymer, polyvinyl chloride, polyethylene, polytetrafluoroethylene, polyurethane, etc.

[0062] The transverse wave velocity of the acoustic resonance immune guide is less than the longitudinal wave velocity of the body fluid (such as blood in the blood vessel) in the area to be tested in the body. The acoustic resonance guide is a hollow or solid linear guide wire. The cross section of the acoustic resonance guide is triangular, circular or rectangular.

[0063] In a preferred embodiment, the circumferential diameter of the acoustic resonance immune guide is 50 μm to 500 μm, and the operating frequency for generating a local strong field mode is 1 MHz to 10 MHz.

[0064] In a preferred embodiment, the material of the acoustic resonance immunoadductor is poly(lactic-co-glycolic acid, PLGA, longitudinal wave velocity 2114 m / s, transverse wave velocity 532 m / s).

[0065] In a preferred embodiment, the acoustic resonance immunoadjuvant may be chemically or biologically modified to increase its adhesion efficiency and targeting. For example, a hydrophilic or hydrophobic coating may be applied to the surface of the acoustic resonance immunoadjuvant as required.

[0066] The capture system in the embodiment of the present invention further includes a microfluidic cell sorting and detection device 50 , and the microfluidic cell sorting and detection device 50 is used to count and detect the cells to be detected captured by the acoustic resonance guide 40 .

[0067] Specifically, the microfluidic cell sorting detection device 50 in the present invention is an ultrasound-based microfluidic cell sorting detection device. Figure 8 As shown, the microfluidic cell sorting and detection device 50 includes: a fluorescent staining pool 51 for cells to be tested, a single cell flow channel 52, an ultrasonic device 53, and a fluorescent microscope imaging system 54. The fluorescent staining pool 51 for cells to be tested is used to fluorescently stain the cells to be tested captured by the in vivo capture system, thereby achieving visibility of the cells to be tested. The single cell flow channel 52 is connected to the fluorescent staining pool 51 for cells to be tested, and the ultrasonic device 53 is arranged outside the channel of the single cell flow channel 52. The ultrasonic focusing wave emitted by the ultrasonic device 53 forms an acoustic field, so that the cell group in the fluorescently stained cell pool to be tested flows through the single cell flow channel 52 and realizes a regular arrangement of single cells, thereby facilitating detection by the downstream fluorescent microscope imaging system 54. The fluorescent microscope imaging system 54 is arranged at the end of the single cell flow channel 52, and is used to identify, count and detect the fluorescently labeled cells to be tested flowing through the single cell flow channel 52.

[0068] The following describes the working method of the in vivo capture system for the cells to be tested, or provides a method for in vivo capture of cells to be tested. Fig. 9 , the method comprises the steps of:

[0069] S10, controlling the immune micro-nano bubble supply device to prepare immune micro-nano bubbles with antibodies that can recognize cells to be tested coupled on the surface, and injecting the immune lipid micro-nano bubbles into the area to be tested in vivo. In this step, after the immune micro-nano bubbles are injected into the area to be tested in vivo, the immune micro-nano bubbles specifically bind to the cells to be tested to form immune micro-nano bubble complexes.

[0070] S20, providing an acoustic resonance immune guide, and determining the operating frequency of the acoustic resonance immune guide. Specifically, based on the diameter and material parameters of the acoustic resonance immune guide, the ultrasonic operating frequency of the local strong field mode generated on its surface is theoretically predicted and experimentally measured. In actual work, the acoustic resonance immune guide can be placed in water, and its operating frequency can be determined by measuring the transmission spectrum to obtain the resonant frequency. In a specific embodiment, the diameter of the PLGA acoustic resonance guide is 300 microns, and the theoretical prediction of its operating frequency of generating a local strong field mode is 1.5MHz.

[0071] S30, controlling the acoustic resonance immunoadhesive body delivery device to deliver the acoustic resonance immunoadhesive body with antibodies coupled to the surface that can recognize the cells to be tested to the area to be tested in the body. In this step, after the acoustic resonance immunoadhesive body is delivered to the area to be tested in the body, the acoustic resonance immunoadhesive body specifically binds to the cells to be tested and directly captures the cells to be tested.

[0072] S40, controlling the ultrasonic emitting device to emit ultrasonic waves to stimulate the acoustic resonance immune guide to generate a local strong field, so that the acoustic resonance immune guide adsorbs the immune micro-nano bubble complex to capture the cells to be tested.

[0073] In this step, first, after the immune micro-nano bubbles and the acoustic resonance immune guide are delivered to the test area in the body for a certain period of time, the ultrasonic transmitting device is controlled to emit ultrasonic waves, wherein the frequency range of the ultrasonic waves emitted by the ultrasonic transmitting device is determined according to the working frequency of the acoustic resonance guide determined in step S20 to meet the resonance requirements.

[0074] S50, controlling the acoustic resonance immune guide body in vivo transport device to transport the acoustic resonance guide body after capturing the cells to be tested to the outside of the body.

[0075] S60, controlling the microfluidic cell sorting detection device to count and detect the cells to be detected captured by the acoustic resonance guide.

[0076] The feasibility of the in vivo capture system and method of cells to be tested provided by the embodiments of the present invention has been verified by numerical simulation.

[0077] Figures 10 to 12 This is the force distribution diagram of immune micro-nano bubbles of different diameters around a PLGA acoustic resonance guide with a diameter of 300 microns and a circular cross-section. The arrow indicates the force direction, and the color represents the force intensity. Fig.10 The diameter of the immune micro-nano bubbles is 2 microns. Fig.11 The diameter of the immune micro-nano bubbles is 4 microns. Fig.12 The diameter of the immune micro-nano bubbles is 6.6 microns. Figures 10 to 12 It can be seen that under the action of the external sound field, the local sound field generated by the resonance of the acoustic resonance guide can make the immune micro-nano bubbles be attracted around the guide body and gather around the guide body, but the particle size of the immune micro-nano bubbles will affect the spatial distribution of the force, so that the bubbles are attracted or repelled. Therefore, it is necessary to prepare immune micro-nano bubbles of suitable particle size by microfluidics, so that as many microbubbles as possible can be captured and attracted, and the diameter of the immune micro-nano bubbles is preferably in the range of 1 μm to 10 μm.

[0078] Fig.13 It is an enlarged photograph under a microscope showing that the cells to be tested are captured by the acoustic resonance guide in an embodiment of the present invention.

[0079] In summary, the in vivo capture system and method of cells to be tested provided in the embodiments of the present invention deliver immune micro-nano bubbles with antibodies coupled to the surface that can recognize cells to be tested and an acoustic resonance immune guide into the area to be tested in the body. On the one hand, the acoustic resonance immune guide can specifically bind to the cells to be tested and directly capture the cells to be tested. On the other hand, under the excitation of ultrasonic waves, the acoustic resonance immune guide wire is used as a "secondary sound source" to generate a local sound field to adsorb and enrich the immune micro-nano bubbles that are combined with the cells to be tested, thereby further capturing a larger number of cells to be tested, thereby increasing the number of trace cells to be tested in the body (such as circulating tumor cells CTC) captured and improving the accuracy of subsequent detection.

[0080] The above is only a specific implementation of the present application. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present application. These improvements and modifications should also be regarded as the scope of protection of the present application.

Claims

1. A system for capturing cells in vivo, characterized in that: It includes an immune micro-nano bubble supply device, an acoustic resonance immune guide body delivery device, an ultrasonic emission device and an acoustic resonance immune guide body; wherein, The immune micro-nano bubble supply device is used to provide immune micro-nano bubbles, the surface of which is coupled with antibodies that can identify cells to be tested, and the immune micro-nano bubbles are used to be injected into the area to be tested in the body and specifically bind to the cells to be tested to form immune micro-nano bubble complexes; The acoustic resonance immune guide body in vivo delivery device is used to deliver the acoustic resonance immune guide body from outside the body to the area to be tested inside the body and from the area to be tested inside the body to outside the body; The ultrasonic emitting device is used to emit ultrasonic waves outside the body to stimulate the acoustic resonance immune guide located inside the body to generate a local strong field; The surface of the acoustic resonance immune guide is coupled with antibodies that can recognize cells to be tested, and is used to specifically bind to cells to be tested and directly capture cells to be tested when being transported to the area to be tested in the body, and to use the local strong field to adsorb the immune micro-nano bubble complex to capture cells to be tested; The immune micro-nano bubble supply device is a microfluidic cavity device, including a primary cavity, a secondary cavity and a tertiary cavity connected in sequence; the primary cavity includes a gas cavity, a liquid cavity, a bubble-forming cavity and an antibody delivery cavity; the secondary cavity is a cavity for coupling and assembling antibodies and micro-nano bubbles; the tertiary cavity is a cavity for screening and obtaining immune micro-nano bubbles with antibodies coupled on the surface that can recognize cells to be tested.

2. The in vivo cell capture system according to claim 1, characterized in that: The diameter of the immune micro-nano bubbles is 1 μm to 10 μm.

3. The in vivo cell capture system according to claim 1, characterized in that: The material of the acoustic resonance immune guide is a flexible material, and the shear wave velocity of the acoustic resonance immune guide is smaller than the longitudinal wave velocity of the body fluid in the area to be tested in the body.

4. The in vivo cell capture system according to claim 3, characterized in that: The material of the acoustic resonance immune guide is a biomedical polymer material.

5. The in vivo cell capture system according to claim 3, characterized in that: The acoustic resonance immune guide body is a hollow or solid linear guide wire, and the cross section of the acoustic resonance immune guide body is a triangle, a circle or a rectangle.

6. The in vivo cell capture system according to any one of claims 3 to 5, characterized in that: The surface of the acoustic resonance immune guide is covered with a hydrophilic or hydrophobic coating, and antibodies capable of identifying cells to be tested are coupled and assembled on the coating.

7. The in vivo cell capture system according to claim 3, characterized in that: The circumferential diameter of the acoustic resonance immune guide is 50 μm to 500 μm, and the working frequency of generating the local strong field mode is 1 MHz to 10 MHz.

8. The in vivo cell capture system according to claim 1, characterized in that: The acoustic resonance immune guide in vivo delivery device is a puncture outer sheath tube, and a pre-installed puncture needle is used to puncture the in vivo test area through the puncture outer sheath tube. The acoustic resonance immune guide is delivered to the internal test area after internal puncture through the puncture outer sheath tube.

9. The in vivo cell capture system according to claim 1, characterized in that: The ultrasonic transmitting device comprises a signal generator, a power amplifier and an ultrasonic transducer. The signal generated by the signal generator is amplified by the power amplifier and then stimulates the ultrasonic transducer to transmit ultrasonic waves.

10. The in vivo cell capture system according to claim 1, characterized in that: The in vivo capture system for cells to be tested also includes a microfluidic cell sorting and detection device for detecting the cells to be tested captured by the acoustic resonance immune guide.

11. The in vivo cell capture system according to claim 1, characterized in that: The cells to be detected are circulating tumor cells, the antibodies capable of recognizing the cells to be detected are antibodies that recognize tumor-specific antigens, and the in vivo region to be detected is a blood vessel.

12. A working method of the in vivo cell capture system according to any one of claims 1 to 11, characterized in that: include: Controlling the immune micro-nano bubble supply device to prepare immune micro-nano bubbles with antibodies coupled on the surface that can recognize cells to be tested, and injecting the immune micro-nano bubbles into the area to be tested in vivo, so that the immune micro-nano bubbles and the cells to be tested specifically bind to form immune micro-nano bubble complexes; Controlling the acoustic resonance immune guide body delivery device to deliver the acoustic resonance immune guide body with antibodies coupled to the surface capable of recognizing the cells to be tested to the area to be tested in the body, so that the acoustic resonance immune guide body specifically binds to the cells to be tested and directly captures the cells to be tested; Controlling the ultrasonic emitting device to emit ultrasonic waves to stimulate the acoustic resonance immune guide to generate a local strong field, so that the acoustic resonance immune guide adsorbs the immune micro-nano bubble complex to capture the cells to be tested; The acoustic resonance immune guide body in vivo delivery device is controlled to deliver the acoustic resonance immune guide body after capturing the cells to be tested to the outside of the body.

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