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WO2021120209A1 - Système et procédé de capture in vivo pour cellule à tester - Google Patents

Système et procédé de capture in vivo pour cellule à tester Download PDF

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Publication number
WO2021120209A1
WO2021120209A1 PCT/CN2019/127154 CN2019127154W WO2021120209A1 WO 2021120209 A1 WO2021120209 A1 WO 2021120209A1 CN 2019127154 W CN2019127154 W CN 2019127154W WO 2021120209 A1 WO2021120209 A1 WO 2021120209A1
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WIPO (PCT)
Prior art keywords
immune
tested
acoustic resonance
cell
cells
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Ceased
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PCT/CN2019/127154
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English (en)
Chinese (zh)
Inventor
郑海荣
徐岗
李飞
毛一雷
孟龙
严飞
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Shenzhen Institute of Advanced Technology of CAS
Peking Union Medical College Hospital Chinese Academy of Medical Sciences
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Shenzhen Institute of Advanced Technology of CAS
Peking Union Medical College Hospital Chinese Academy of Medical Sciences
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Publication of WO2021120209A1 publication Critical patent/WO2021120209A1/fr
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B10/00—Instruments for taking body samples for diagnostic purposes; Other methods or instruments for diagnosis, e.g. for vaccination diagnosis, sex determination or ovulation-period determination; Throat striking implements
    • A61B10/02—Instruments for taking cell samples or for biopsy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/145—Measuring characteristics of blood in vivo, e.g. gas concentration or pH-value ; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid or cerebral tissue
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses

Definitions

  • the invention belongs to the technical field of cell detection in vivo, and particularly relates to a system and method for capturing cells to be tested in vivo.
  • Circulating tumor cell is an important medium for tumor occurrence and metastasis.
  • CTC Circulating tumor cell
  • CTC will be released into the blood circulation by the tumor or metastasis at the primary site, thereby colonizing the distant tissues or organs of the body.
  • the formation of new metastatic lesions As a safe and reliable biomarker, CTC has the advantages of non-invasiveness, easy access, and high reproducibility. Its application in the field of malignant tumors is developing rapidly. Related research covers early tumor diagnosis, treatment, prognostic evaluation, and pathogenesis. field.
  • the blood of cancer patients may contain hundreds or thousands of CTCs
  • the traditional detection method of 5-10 ml blood samples usually contains only a small amount of CTC, this order of magnitude Tumor cells are not sufficient for comprehensive detection of biological behaviors such as tumor molecular biology heterogeneity and drug resistance mutations.
  • a low number of CTCs may lead to false-negative results of tumor blood samples, especially in early tumor detection or tumor recurrence detection, leading to misdirection of clinical decision-making, resulting in irreversible loss of treatment and survival prognosis of tumor patients.
  • CTC detection technologies can be divided into three main categories, namely: CTC detection methods based on nucleic acid expression, CTC detection methods based on cell physical characteristics, and CTC detection methods based on cell surface antibodies .
  • CTC CTC by nucleic acid expression
  • CTC CTC
  • blood component cells RNA expression of CTC which is different from blood component cells
  • the nucleic acids carried by the cells can significantly affect the test results, which leads to obvious drawbacks of this technology. It is currently believed that compared with blood component cells, CTC has a larger volume and density, and the cell surface charge distribution is also different from other cells. In addition, some CTCs may also exhibit different cell activities and other characteristics. These specific differences have gradually been Recognize and apply it to cell detection and screening.
  • Epithelial cell adhesion molecule is widely used in CTC detection based on cell surface antibodies because it is expressed in almost all epithelial-derived cells but not in blood component cells.
  • Epithelial tumor cells are enriched and combined with immunomagnetic beads coupled with a specific anti-EpCAM antibody, and then screened by a specific magnetic field. They have been used for the detection of cancer CTCs such as lung cancer, breast cancer and colorectal cancer.
  • cancer CTCs such as lung cancer, breast cancer and colorectal cancer.
  • two representative CTC detection technologies developed based on this principle, namely the U.S.
  • FDA Food and Drug Administration
  • CFDA China Food and Drug Administration
  • the CellSearch system a commercial product produced by Veridex, and the only CellCollector sampling needle produced by Gilupi in Germany that captures CTC in the body and is approved by the CFDA.
  • the CellSearch system is a relatively standard CTC detection method among the existing detection technology methods and has begun to be used in some clinical trials, the main disadvantage of the system is its low sensitivity (approximately only 1 CTC cell per milliliter of blood is detected) And a single test usually requires a larger blood sample, and the effective detection of CTC can be achieved only in the peripheral blood of patients with distant metastasis of the tumor. See: Andree, KC; van Dalum, G.; Terstappen, LWChallenges in circulating tumor cell detection by the CellSearch system. Mol Oncol 2016; 10: 395-407.
  • CellCollector sampling needle related research proves that it has more advantages in CTC detection cell volume acquisition and sensitivity.
  • CellCollector sampling is limited in its ability to capture CTC in vivo. It has a poor ability to attract and capture CTC cells flowing in blood vessels. 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.
  • the present invention provides a system for capturing cells to be tested in vivo and a working method thereof, which are used to increase the number of trace cells to be tested in vivo (such as circulating tumor cell CTCs) and improve the accuracy of subsequent detection. degree.
  • An in-vivo capture system for cells to be tested comprising an immune micro-nano bubble supply device, an acoustic resonance immune guide body conveying device, an ultrasonic emission device and an acoustic resonance immune guide body;
  • the immune micro/nano bubble supply device is used to provide immune micro/nano bubbles, the surface of the immune micro/nano bubbles is coupled with antibodies capable of identifying cells to be tested, and the immune micro/nano bubbles are used to inject into the area to be tested in the body. Specific binding with the cell to be tested to form an immune micro-nano bubble complex;
  • the device for delivering the acoustic resonance immune guide body in vivo is used to transport the acoustic resonance immune guide body from the outside of the body to the area to be tested in the body and from the area to be tested in the body to the outside of the body;
  • the ultrasonic transmitting device is used for transmitting ultrasonic waves outside the body to excite the acoustic resonance immune guide body located in the body to generate a local strong field;
  • the surface of the acoustic resonance immune guide is coupled with an antibody capable of recognizing the cell to be tested, and is used to specifically bind to the cell to be tested when it is delivered to the area to be tested in the body to directly capture the cell to be tested, and use all the cells to be tested.
  • the local strong field adsorbs the immune micro-nano bubble complex to capture the cells to be tested.
  • the immunological micro-nano bubble supply device is a microfluidic cavity device, which includes a first-level cavity, a second-level cavity, and a third-level cavity that are sequentially connected;
  • the first-level cavity includes a gas cavity, a liquid grease Plasma cavity, bubble formation cavity, and antibody delivery cavity;
  • the secondary cavity is a cavity for coupling and assembly of antibodies and micro-nano bubbles;
  • the tertiary cavity is a screening and acquisition surface coupled with an identification to be tested Cells are immune to the cavities of the micro-nano bubbles.
  • the diameter of the immune micro-nano bubbles is 1 ⁇ m-10 ⁇ m.
  • the material of the acoustic resonance immune guide is a flexible material; the transverse wave velocity of the acoustic resonance immune guide is smaller than the longitudinal wave velocity of the body fluid in the area to be measured in the body.
  • the material of the acoustic resonance immune guide is a biomedical polymer material.
  • the acoustic resonance immune guide is a hollow or solid linear guide wire; the cross section of the acoustic resonance immune guide is triangular, circular or rectangular.
  • the surface of the acoustic resonance immune guide is covered with a hydrophilic or hydrophobic coating, and antibodies capable of recognizing the cells to be tested are coupled and assembled on the coating.
  • the circumferential diameter of the acoustic resonance immune guide is 50 ⁇ m to 500 ⁇ m, and the working frequency for generating the local strong field mode is 1 MHz to 10 MHz.
  • the acoustic resonance immune guide body delivery device is a puncture outer sheath, and a pre-installed puncture needle is used to puncture the area to be tested in the body through the puncture outer sheath, and the acoustic resonance immune guide body It is delivered to the inner area to be tested after the inner puncture through the puncture outer sheath.
  • the ultrasonic transmitting device includes a signal generator, a power amplifier, and an ultrasonic transducer, and the signal generated by the signal generator is amplified by the power amplifier to excite the ultrasonic transducer to emit ultrasonic waves.
  • the system for capturing cells to be tested in vivo also includes a microfluidic cell sorting and detection device for detecting cells to be tested captured by the acoustic resonance immune inducer.
  • the test cell is a circulating tumor cell
  • the antibody capable of recognizing the test cell is an antibody that recognizes a tumor-specific antigen
  • the test area in the body is a blood vessel.
  • Another aspect of the present invention provides a working method of the in vivo capture system for the cells to be tested as described above, which includes:
  • the immune micro/nano bubble supply device is controlled to prepare and obtain immune micro/nano bubbles with antibodies capable of identifying cells to be tested on the surface, and the immune micro/nano bubbles are injected into the area to be tested in the body, so that the immune micro/nano bubbles are in contact with the cells to be tested.
  • the test cells specifically bind to form an immune micro-nano bubble complex
  • Control the acoustic resonance immune guide body delivery device to transport the acoustic resonance immune guide body coupled with an antibody capable of recognizing the cell to be tested to the area to be tested in the body, so that the acoustic resonance immune guide body and the cell to be tested Specific binding occurs directly to capture the cell to be tested;
  • the acoustic resonance immune inducer in vivo delivery device is controlled to transport the acoustic resonance immune inducer after capturing the cells to be tested to the outside of the body.
  • the immune micro-nano bubble with an antibody capable of recognizing the test cell and the acoustic resonance immune guide are coupled to the area to be detected in the body, and the acoustic resonance immune guide
  • the primer can specifically bind to the cell to be tested and directly capture the cell to be tested.
  • the acoustic resonance immune guide is used as a "secondary sound source" to generate a local sound field for adsorption and enrichment.
  • the immune micro-nano bubbles combined with the cells to be tested can further capture a larger number of cells to be tested, increase the number of trace cells to be tested in the body (such as circulating tumor cells CTC), and improve the accuracy of subsequent detection.
  • the present invention can continuously recruit and capture CTC during the continuous blood circulation in the body, thereby continuously increasing the local concentration of CTC cells, and can capture a larger number of CTCs. cell.
  • the present invention can not only capture the CTC cells flowing through the surface of the acoustic resonance immune guide body, but also can guide the resonance immunity through the adsorption force of the strong local field. CTC cells within a certain distance around the primer can be captured, which can capture a larger number of CTC cells.
  • ultrasound-controlled particles is a non-contact, non-invasive, and convenient method of manipulation, which uses sound manipulation to immune micro-nano bubbles
  • the process of capturing CTC will not cause cell damage and ensure the biological activity of CTC, that is, the in vivo capture system and working method of the cell to be tested provided by the present invention are safer and more reliable.
  • FIG. 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
  • Fig. 2 is a diagram of an immune micro-nano bubble coupled with an antibody capable of recognizing the cell to be tested in an embodiment of the present invention
  • FIG. 3 is a diagram showing the combination of immune micro-nano bubble and test cell to form an immune micro-nano bubble complex in an embodiment of the present invention
  • FIG. 4 is a schematic diagram of the structure of an immune micro-nano bubble supply device in an embodiment of the present invention.
  • FIG. 5 is an exemplary diagram of the structure of the acoustic resonance immune guide body delivery device in the embodiment of the present invention and the process of delivering the acoustic resonance immune guide body;
  • Fig. 6 is a schematic diagram of the structure of an ultrasonic transmitting device in an embodiment of the present invention.
  • Fig. 7 is a schematic structural diagram of an acoustic resonance immune inducer in an embodiment of the present invention.
  • Fig. 8 is a schematic structural diagram of a microfluidic cell sorting and detection device according to an embodiment of the present invention.
  • FIG. 9 is a working flow chart of the method for capturing cells to be tested in vivo according to an embodiment of the present invention.
  • Figures 10 to 12 are diagrams showing the force distribution of immune micro-nano bubbles of different sizes and diameters around an acoustic resonance guide with a circular cross-section of 300 microns in diameter;
  • Fig. 13 is a diagram showing an enlarged photograph of a cell to be tested captured by an acoustic resonance guide in an embodiment of the present invention.
  • FIG. 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.
  • the capture system for cells to be tested in vivo includes: an immune micro-nano bubble supply device 10 and an acoustic resonance immune guide.
  • the capture system of this embodiment further includes a microfluidic cell sorting detection device 50.
  • the immune micro/nano bubble supply device 10 is used to provide immune micro/nano bubbles. 2 and 3, the surface of the immune micro/nanobubble 1 is coupled with an antibody 2 capable of identifying the cell to be tested, and the immune micro/nanobubble 1 is used to be injected into the area to be tested in the body and interact with the cell to be tested 3. Specific binding to form an immune micro-nano bubble complex.
  • the test cell 3 is, for example, a circulating tumor cell (CTC)
  • the antibody 2 that can recognize the test cell is an antibody that recognizes a tumor-specific antigen
  • the test area in the body refers to Blood vessels.
  • the immune micro-nano bubble supply device 10 is a microfluidic channel device, as shown in FIG. 4, the microfluidic channel device includes a primary channel 11, a secondary channel 12 and Three-level cavity 13.
  • the primary cavity 11 includes a gas cavity 11a, a liquid lipid cavity 11b, a bubble formation cavity 11c, and an antibody delivery cavity 11d.
  • the gas cavity 11a is used to supply the inert gas G, such as perfluoropropane, which forms the core of the immune micro/nano bubble;
  • the liquid lipid cavity 11b is used to supply the liquid lipid material L that forms the shell of the immune micro/nano bubble, For example, phospholipids;
  • the bubble-forming cavity 11c is connected to the output ends of the gas cavity 11a and the liquid lipid cavity 11b, and the bubble-forming cavity 11c is a combination of inert gas G and liquid lipid material L to form immune micro-nano bubbles
  • the cavity of 1; the antibody delivery cavity 11d is used to supply antibodies 2 capable of recognizing the cells to be tested.
  • the secondary cavity 12 is connected to the output end of the bubble formation cavity 11c and the antibody delivery cavity 11d, and the secondary cavity 12 is a cavity where the antibody 2 and the immune micro/nano bubble 1 are coupled and assembled, and the two An ultrasound device 14 is arranged outside the end of the stage cavity 12, and the immune micro/nano bubbles equipped with antibodies are sorted by transmitting ultrasonic waves 15.
  • the tertiary cavity 13 is connected to the output end of the secondary cavity 12, and the tertiary cavity 13 is a cavity for screening and acquiring immune micro-nano bubbles with antibodies capable of recognizing the cells to be tested on the surface.
  • the three-stage cavity 13 is used for screening and removing immune micro-nano bubbles 1 with uniform particle size.
  • microfluidic cavity device further includes an excretion channel 16 for discharging antibodies that have not been coupled and assembled, micro-nano bubbles, and immune micro-nano bubbles that are coupled to antibodies with a particle size that does not meet the requirements.
  • the micro-bubbles produced by the fluid control channel device of the present invention can achieve uniform and single particle size, thereby greatly optimizing the trapping efficiency of the micro-nano bubbles in the body.
  • the immune micro/nano bubble provided by the embodiment of the present invention is a lipid immune micro/nano bubble, which includes a lipid shell coupled with an antibody capable of recognizing the cell to be tested and an inert gas inner core, and its diameter is preferably in the range of 1 ⁇ m to 10 ⁇ m .
  • the immune micro-nano bubbles can also be chemically or biologically modified to increase their adhesion efficiency and targeting.
  • the acoustic resonance immune guide body delivery device 20 is used to transport the acoustic resonance immune guide body 40 from the outside of the body to the area to be tested in the body, or from the area to be tested in the body to the outside of the body.
  • the acoustic resonance immune guide body delivery device 20 is specifically a puncture outer sheath 4, which is equipped with a puncture needle 5.
  • the puncture outer sheath 4 includes a puncture needle port 4a, an introducer port 4b, and a puncture port 4c.
  • the process of delivering the acoustic resonance immune guide body 40 into the body by the acoustic resonance immune guide body delivery device 20 is as follows: firstly, the puncture needle 5 is put into the puncture outer sheath 4 from the puncture needle port 4a, and the puncture port 4c is aligned The quasi-target area (for example, blood vessel) is punctured.
  • the quasi-target area for example, blood vessel
  • the puncture needle 5 is withdrawn, and at this time, the acoustic resonance immune introducer 40 is put into the puncture outer sheath 4 from the introducer port 4b, and sent through the puncture port 4c. Enter the target area after being pierced.
  • the ultrasonic transmitting device 30 is used for transmitting ultrasonic waves outside the body to excite the acoustic resonance immune guide body 40 in the body to generate a local strong field.
  • 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 excite The ultrasonic transducer 33 emits ultrasonic waves.
  • the ultrasonic transducer 33 may be one of a single-element ultrasonic transducer, a phased-array ultrasonic transducer, a linear-array ultrasonic transducer, and a convex-array ultrasonic transducer; among them, the acoustic resonance immune guidance
  • the resonance frequency of the body 40 determines the driving frequency of the transmitted ultrasound, and thus determines the center frequency of the ultrasonic transducer 33.
  • the signal generated by the signal generator 31 may be a continuous sinusoidal signal or a pulsed sinusoidal signal.
  • the signal generator 31 may be a programmable signal generator (AFG3021, Tectronix), and the power amplifier may be a 50dB linear power amplifier (325LA, ENI), signal generation, 31 generates a sinusoidal continuous signal, a sinusoidal signal After the power amplifier 32, the ultrasonic transducer 33 is excited to generate ultrasonic waves.
  • AFG3021, Tectronix programmable signal generator
  • the power amplifier may be a 50dB linear power amplifier (325LA, ENI)
  • signal generation 31 generates a sinusoidal continuous signal
  • a sinusoidal signal After the power amplifier 32, the ultrasonic transducer 33 is excited to generate ultrasonic waves.
  • the surface of the acoustic resonance immune guide 40 is coupled with an antibody 41 capable of recognizing the cell to be tested, and at least the part delivered to the target area is coupled with an antibody 41 capable of recognizing the cell to be tested; Further, in this embodiment, the surface of the acoustic resonance immune guide 40 is first covered with a hydrophilic or hydrophobic coating 44, and an antibody 41 capable of identifying the cells to be tested is coupled and assembled on the coating 44.
  • the acoustic resonance immune guide body 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 test cell to directly capture the test cell when it is transported to the test area in the body, and use the local strong field to adsorb the immune micro/nano bubbles Complex to capture the cells to be tested.
  • the acoustic resonance immune introducer 40 when the acoustic resonance immune introducer 40 is inserted into the puncture outer sheath 4 from the introducer port 4b, the placement mark 42 of the acoustic resonance immune introducer 40 is sent to and guided The pull-up port 4b is flush. After the acoustic resonance immune guide 40 captures the cells to be tested, the acoustic resonance immune guide 40 is slowly withdrawn from the introducer port 4b until the exit mark 43 is flush with the introducer port 4b, and then the entire puncture The sheath 4 is withdrawn from the target area.
  • the material of the acoustic resonance immune guide is a flexible material, and is a biomedical polymer material, such as polylactic acid-glycolic acid copolymer, polyvinyl chloride, polyethylene, polytetrafluoroethylene, polyurethane, etc.
  • the transverse wave velocity of the acoustic resonance immune guide is smaller than the longitudinal wave velocity of the body fluid (for example, blood in the blood vessel) in the area to be measured in the body.
  • the acoustic resonance guide body is a hollow or solid linear guide wire.
  • the cross section of the acoustic resonance guide is triangular, circular or rectangular.
  • the circumferential diameter of the acoustic resonance immune guide body is 50 ⁇ m to 500 ⁇ m, and the working frequency for generating the local strong field mode is 1 MHz to 10 MHz.
  • the material of the acoustic resonance immune guide is poly(lactic-co-glycolic acid) (PLGA, longitudinal wave velocity 2114 m/s, transverse wave velocity 532 m/s).
  • the acoustic resonance immune inducer can also be chemically or biologically modified to increase its adhesion efficiency and targeting.
  • the surface of the acoustic resonance immune guide is covered with a hydrophilic or hydrophobic coating.
  • the capture system in the embodiment of the present invention further includes a microfluidic cell sorting and detection device 50, the microfluidic cell sorting and detection device 50 is used to count the cells to be tested captured by the acoustic resonance guide 40 And detection.
  • the microfluidic cell sorting detection device 50 in the present invention is a microfluidic cell sorting detection device based on ultrasound.
  • 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 ultrasound device 53 and a fluorescence microscope imaging system 54.
  • the fluorescent staining pool 51 for the cells to be tested is used for fluorescently staining the cells to be tested captured by the in vivo capture system, so as to realize the visibility of the cells to be tested.
  • the single-cell circulation channel 52 is connected to the fluorescent staining pool 51 for the cells to be tested, and the ultrasound device 53 is arranged outside the cavity of the single-cell circulation channel 52, and the ultrasonic focused wave emitted by the ultrasound device 53 forms a sound field, so that The cell population in the cell pool to be tested that is fluorescently stained flows through the single cell flow channel 52 and realizes the regular arrangement of the single cells, thereby facilitating the detection by the downstream fluorescence microscope imaging system 54.
  • the fluorescence microscope imaging system 54 is arranged at the end of the single-cell circulation channel 52 for identifying, counting and detecting the fluorescently labeled cells to be tested flowing through the single-cell circulation channel 52.
  • the method includes the steps:
  • Control the immune micro-nano bubble supply device to prepare and obtain immune micro-nano bubbles with antibodies capable of identifying cells to be tested on the surface, and inject the immuno-lipid micro-nano bubbles into the area to be tested in the body.
  • the immune micro/nano bubbles specifically bind with the cells to be tested to form an immune micro/nano bubble complex.
  • S20 Provide an acoustic resonance immune inducer, and determine the working frequency of the acoustic resonance immune inducer.
  • the acoustic resonance immune guide body can be placed in water, and its operating frequency can be determined by measuring the transmission spectrum to obtain the resonance frequency.
  • the diameter of the PLGA acoustic resonance guide is 300 microns, and theoretically predicts that its working frequency for generating a local strong field mode is 1.5 MHz.
  • the micro-nano bubbles to be immune and the acoustic resonance immune guide are delivered to the area to be measured in the body for a certain period of time, and then the ultrasonic transmitter is controlled to emit ultrasonic waves, and the acoustic resonance guide determined in step S20 is required.
  • the working frequency of the body determines the frequency range of the ultrasonic waves emitted by the ultrasonic transmitter so as to meet the requirements of resonance.
  • Figures 10 to 12 are diagrams showing the force distribution of immune micro-nano bubbles of different sizes and diameters around a PLGA acoustic resonance guide with a diameter of 300 microns and a circular cross-section. Among them, the arrow indicates the direction of the force, and the color represents the strength of the force.
  • the diameter of the immune micro/nano bubbles in Figure 10 is 2 microns
  • the diameter of the immune micro/nano bubbles in Figure 11 is 4 microns
  • the diameter of the immune micro/nano bubbles in Figure 12 is 6.6 microns. It can be seen from Fig. 10 to Fig.
  • the local sound field generated by the resonance of the acoustic resonance guide body can make the immune micro-nano bubbles be attracted around the guide body and gather around the guide body.
  • the particle size of the immune micro-nano bubbles will affect the spatial distribution of the force, causing the bubbles to be attracted or repelled. Therefore, it is necessary to prepare immune micro/nano bubbles of suitable particle size by a microfluidic method, so as to capture and attract as many microbubbles as possible.
  • the diameter of the immune micro/nano bubbles is preferably in the range of 1 ⁇ m to 10 ⁇ m.
  • FIG. 13 is an illustration of a magnified photograph under a microscope in which the acoustic resonance guide captures the cell to be tested in an embodiment of the present invention.
  • the system and method for capturing cells to be tested in vivo are delivered into the body to be tested by coupling the immune micro-nano bubbles with antibodies capable of recognizing the cells to be tested and the acoustic resonance immune guide.
  • the acoustic resonance immune guide can specifically bind to the cell to be tested and directly capture the cell to be tested.
  • the acoustic resonance immune guide wire is used as the "secondary" to generate the local sound field.
  • the “infrasound source” adsorbs and enriches the immune micro-nano bubbles combined with the cells to be tested, and further captures a larger number of cells to be tested, increases the number of trace cells to be tested in the body (such as circulating tumor cells CTC), and improves subsequent detection Accuracy.

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Abstract

L'invention concerne un système de capture in vivo pour une cellule à tester et un procédé de fonctionnement de celui-ci. Le système comprend un dispositif d'alimentation en micro-nano-bulles immunitaires (10), un dispositif de transport in vivo de corps de guidage immunitaire à résonance acoustique (20), un dispositif de transmission ultrasonore (30), et un corps de guidage immunitaire à résonance acoustique (40), le dispositif d'alimentation en micro-nano-bulles immunitaires (10) fournissant une micro-nano-bulle immunitaire couplée à un anticorps, et la micro-nano-bulle immunitaire étant injectée dans un corps pour être combinée spécifiquement à ladite cellule afin de former un complexe de bulles ; le dispositif de transport in vivo de corps de guidage (20) transporte le corps de guidage immunitaire à résonance acoustique (40) dans une zone in vivo à tester ; le dispositif de transmission ultrasonore (30) transmet une onde ultrasonore pour exciter le corps de guidage immunitaire à résonance acoustique (40) in vivo afin de générer un champ local fort ; et la surface du corps de guidage immunitaire à résonance acoustique (40) est couplée à un anticorps capable de reconnaître ladite cellule, le corps de guidage immunitaire à résonance acoustique est spécifiquement combiné à ladite cellule pour capturer ladite cellule lorsqu'il est transporté dans le corps, et le champ local fort est utilisé pour adsorber le complexe de bulles afin de capturer ladite cellule. Le système peut augmenter la quantité de capture de cellules de trace à tester in vivo, et améliorer la précision de tests ultérieurs.
PCT/CN2019/127154 2019-12-18 2019-12-20 Système et procédé de capture in vivo pour cellule à tester Ceased WO2021120209A1 (fr)

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