WO2019196270A1 - 微纳粒子检测系统及方法 - Google Patents

微纳粒子检测系统及方法 Download PDF

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WO2019196270A1
WO2019196270A1 PCT/CN2018/098650 CN2018098650W WO2019196270A1 WO 2019196270 A1 WO2019196270 A1 WO 2019196270A1 CN 2018098650 W CN2018098650 W CN 2018098650W WO 2019196270 A1 WO2019196270 A1 WO 2019196270A1
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micro
sample chamber
chamber unit
sample
nano particles
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English (en)
French (fr)
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孙佳姝
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国家纳米科学中心
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Priority to US17/043,038 priority Critical patent/US20210033504A1/en
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G01N1/4022—Concentrating samples by thermal techniques; Phase changes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G01N1/4077—Concentrating samples by other techniques involving separation of suspended solids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/44—Sample treatment involving radiation, e.g. heat
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/06—Investigating concentration of particle suspensions
    • G01N15/0606—Investigating concentration of particle suspensions by collecting particles on a support
    • G01N15/0612—Optical scan of the deposits
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6402—Atomic fluorescence; Laser induced fluorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/01—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials specially adapted for biological cells, e.g. blood cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N2015/0038—Investigating nanoparticles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks

Definitions

  • the invention relates to the field of micro-nano particle detection technology, in particular to a micro-nano particle detection system and method based on a thermophoretic effect.
  • micro-nano particles are detected to measure particle size, shape, concentration, activity, etc., and are widely used in hematology, immunology, molecular biology, clinical medicine and other disciplines.
  • the flow particle detection method is often used to detect micro-nano particles, which is a technique for quantitative analysis and sorting of particle particles in a liquid one by one.
  • the Coulter principle used in the detection refers to: suspension When the particles in the electrolyte pass through the small holes, the same volume of electrolyte is replaced. In the circuit of constant current design, the resistance between the two electrodes inside and outside the small hole changes instantaneously, and the potential pulse and the magnitude and frequency of the pulse signal are generated. It is proportional to the size and number of particles.
  • Sample focusing is the key technology for flow particle detection. In the current testing, the sample liquid is focused by external force. Focusing is further divided into focusing through the sheath fluid and focusing without the sheath fluid.
  • the sheath liquid is focused as in the "microfluidic microparticle instrument and manufacturing method" disclosed in Chinese Patent No. 201210482142.7, and the sample liquid is injected from the sample liquid inlet by the pressure of the external syringe pump, the sheath liquid is injected from the sheath liquid inlet, and then the sample liquid and the two
  • the sheath fluid flows simultaneously to the sheath flow converging zone, and the aggregation of the sheath fluid sandwiches the particulate particles in the sample liquid into a linear arrangement and flows into the detection zone for detection.
  • both the sheath flow and the sample solution require a drive source, and a motor is used to control the three pipes, so that not only the equipment becomes large, the cost is increased, and more importantly, it is required for each test.
  • the chip is replaced, the three channels need to be reconnected to the motor every time. The sealing problem at this joint will affect the pressure on the three channels, resulting in poor focusing and insufficient test results. accurate.
  • the nanoparticles are separated and detected by an electrochemical method to form a stream containing micro-nano particles, and the amount of samples required is extremely large;
  • a driving source such as a motor, and a single channel of a fixed structure, the flow direction and the accumulating direction of the micro-nano particles are defined, and in the process of applying the external force and the channel defining, the external force acts on the fluid, often targeting The force applied to the micro-nano particles is uncontrollable.
  • exosomes are secreted by cells, used for intercellular communication, because they contain proteins and genetic material related to the mother cells, which can regulate a variety of physiology or Pathological reactions, including tumor cell invasion and metastasis, blood vessel growth, immune response, etc., have gradually become an emerging biomarker for non-invasive tumor diagnosis in recent years.
  • the use of exosomes for tumor diagnosis often requires analysis of their surface protein types, but due to the lack of accurate, feasible and easy-to-use analytical methods, there are still challenges in analyzing the small differences in protein on different exosomes.
  • an enzyme-linked immunosorbent assay that is, an ELISA
  • an enzyme-linked immunosorbent assay refers to a qualitative and quantitative detection method in which a soluble antibody is bound to a solid phase carrier such as polystyrene, and an antigen-antibody binding specific immunological reaction is utilized.
  • the test specimen the antibody in the assay
  • the enzyme-labeled antibody are reacted with the antigen on the surface of the solid phase carrier in different steps; the antigen-antibody complex formed on the solid phase carrier is washed and other methods.
  • the substances are separated, and finally the amount of enzyme bound to the solid support is proportional to the amount of the test substance in the specimen.
  • the substrate of the enzymatic reaction After the substrate of the enzymatic reaction is added, the substrate is catalyzed by the enzyme to become a colored product, and the amount of the product is directly related to the amount of the test substance in the sample, so that it can be qualitatively or quantitatively analyzed according to the depth of the color reaction.
  • Western Blot the basic principle of which is to color the gel-electrophoresis-treated cells or biological tissue samples by specific antibodies; to obtain specific proteins in the cells analyzed by analyzing the position and depth of staining. Or information in the organization that expresses the situation.
  • the present invention provides a micro/nano particle detecting system including a heating unit and a sample chamber unit, wherein
  • the heating unit is configured to heat a sample in the sample chamber unit
  • the sample chamber unit is loaded with micro-nano particle fluid, and after the heating unit heats the sample chamber unit, a thermophoretic effect is generated in the sample chamber unit to concentrate the micro-nano particles in the The temperature in the sample chamber unit is lower than the side of the micro-nano particle fluid for detection.
  • system further includes a signal acquisition unit, and the signal acquisition unit collects related information of the aggregated micro-nano particles, and performs corresponding analysis.
  • the sample chamber unit includes a sealed sample chamber for loading the micro/nanoparticle fluid and for providing a space for generating a thermophoresis effect, the sample chamber comprising: blocking the sample chamber and accumulating the micro/nano particles a second heat conducting surface, the temperature near the second heat conducting surface is lower than the temperature of the micro/nano particle fluid to generate a temperature difference between the second heat conducting surface and the micro/nano particle fluid, generating a thermophoretic effect, driving the micro The nanoparticles are oriented to move toward the second heat transfer surface.
  • the heating unit is a laser that illuminates the sample chamber unit, and the light beam sequentially passes through the micro/nano particle fluid and the second heat conducting surface to generate a thermophoretic effect on the micro/nano particle solution.
  • the sample chamber further includes: a first heat conducting surface for closing the sample chamber, wherein the second heat conducting surface and the first heat conducting surface both pass the light beam.
  • the second heat conducting surface is a transparent material, which is made of sapphire; and the first heat conducting surface is any one of glass, polymethyl methacrylate, polydimethylsiloxane, sapphire, and diamond. Or a combination of any of several.
  • micro/nanoparticles are exosomes, extracellular vesicles, cells or microspheres with good biocompatibility.
  • micro-nano particles are immuno-microspheres to which a target biomolecule is bound, and the immuno-microspheres are prepared by immobilizing an antibody or an aptamer on the surface of the microsphere.
  • the invention also provides a method for detecting micro-nano particles, comprising: heating a fluorescently-labeled micro-nano particle fluid in a sample chamber unit, generating a temperature difference in the sample chamber unit to be single in the sample chamber Generating a thermophoretic effect to concentrate fluorescently labeled micro-nanoparticles on a side of the sample chamber unit temperature below the micro-nanoparticle fluid to amplify the labeled fluorescent signal;
  • step b the corresponding index information of the micro-nano particles is collected by analyzing the micro-nano particles accumulated on the low temperature side in the sample chamber unit, and the corresponding indexes are analyzed.
  • micro/nanoparticles are exosomes or immunoglobulins bound with a target biomolecule, and the immunoglobulin is prepared by immobilizing an antibody or an aptamer on the surface of the microsphere.
  • the invention has the beneficial effects that the micro-nano detection system of the invention heats one of the sample chamber units in which the micro-nano particles are located, introduces a thermophoretic effect and convection, and generates a sample chamber unit.
  • the temperature difference generates a low temperature on the side away from the heating unit, and the thermophoretic effect causes the micro-nano particles in the sample to migrate and accumulate to the sample chamber unit to complete the accumulation of the micro-nano particles; meanwhile, the sample liquid is thermally expanded to generate buoyancy and thus the sample Convection occurs in the chamber unit.
  • the direction of convection is directed from the periphery to the heating region of the sample chamber unit, further accelerating the accumulation of micro-nano particles.
  • the lower surface of the sample chamber is designed as a highly transparent material that causes the exosomes to migrate to the lower surface of the lower temperature sample chamber.
  • convection can accelerate and enhance the concentration of exosomes, thereby increasing the signal magnification.
  • the system incubated the test sample containing the exosomes with the fluorescently labeled aptamer or antibody, and the exosomes are labeled with fluorescence by specific binding of the aptamer or antibody to the exosome surface protein;
  • the sample is placed in a transparent sample chamber and placed on a fluorescence microscope stage for observation.
  • the infrared laser irradiates through the sample chamber to act on the sample, and the exosome in the sample is highly enriched in the bottom of the sample chamber by thermophoresis.
  • the exosome fluorescence is highly amplified, and the abundance of a certain exosome surface protein is detected by the fluorescence intensity.
  • the system uses a laser to heat the sample chamber, and a transparent heat conduction surface having different thermal conductivity is disposed on the opposite side of the sample chamber to generate a temperature difference between the two heat conducting surfaces to generate a thermophoretic effect, thereby driving the micro/nano particles from
  • the second heat conducting surface of the first heat conducting surface having a low temperature is oriented to move.
  • the use of beam heating does not require the use of other auxiliary equipment, as long as the transparent heat transfer surface is placed above and below the sample chamber.
  • the force of the micro/nano particles is proportional to the square of the particle diameter under the thermophoretic effect, and is independent of the number of micro-nano particles.
  • micro-nano particles can be used for aggregation and detection, and only for exosomes 0.1 microliter sample volume, easy to operate, no special equipment, no sample preparation and exosome purification, common to aptamers and antibodies; not limited to exosomes, other extracellular vesicles, cells and other micro-nano biological particles Yes.
  • the micro/nanoparticle detection system and method of the present invention can select a measurement at a specific temperature, is not limited by a specific temperature, and can only generate a temperature difference to accumulate particles; it can also be completed in various solution environments. Measurements, including the complex detergent environment required to study membrane proteins; can also detect molecules of various sizes: ions, nucleic acid fragments, nucleosomes, liposomes, when specifically tested, the system can According to the physical properties of the particles and the size of the particles, adjust the temperature difference, the height between the upper and lower heat transfer surfaces, the type of fluid and the frequency of laser irradiation, etc., and adjust the above parameters to achieve quantitative adjustment and precise control. Easy to adjust.
  • biomacromolecules such as free proteins and nucleic acids of the present invention or biological macromolecules such as proteins and nucleic acids which are not exposed on the surface of the exosomes, and the antibodies or aptamers which specifically bind to the target protein and nucleic acid are modified on the surface of the micron-sized spheres.
  • the invention accumulates particles based on the thermophoresis effect, and the loading container of the micro/nano particles is not limited, especially in a container with a large volume, the particles are more likely to accumulate under the thermophoretic effect without considering the use of a carrier container such as a capillary for guiding.
  • FIG. 1 is a structural block diagram of a micro/nano particle detecting system of the present invention
  • FIG. 2 is a block diagram of an exosome-based signal detection process of the present invention.
  • Example 3 is a comparative map of exosomes of Example 1 of the present invention before and after the test;
  • Example 5 is a schematic diagram showing the expression levels of various proteins in serum exosomes of various cancer patients in healthy individuals according to Example 2 of the present invention.
  • FIG. 6 is a schematic diagram showing fluorescence measurement gradation values of fluorescent polystyrene microspheres of different diameters according to Example 3 of the present invention.
  • Figure 7 is a schematic diagram showing the expression levels of 11 protein markers in serum of ovarian cancer patients and healthy humans according to Example 4 of the present invention.
  • Fig. 8 is a schematic view showing the correct rate of 11 different markers and their sums as a difference in cancer and health standards according to Example 4 of the present invention.
  • the terms “installation”, “connected”, and “connected” are to be understood broadly, and may be fixed connections, for example, or It is a detachable connection, or an integral connection; it may be a mechanical connection or an electrical connection; it may be directly connected or indirectly connected through an intermediate medium, and may be internal communication between the two elements.
  • the specific meanings of the above terms in the present invention can be understood on a case-by-case basis.
  • FIG. 1 is a structural block diagram of the micro/nano particle detecting system of the present invention.
  • the system of the embodiment includes a heating unit 1 , a sample chamber unit 2 and a signal collecting unit 4 , wherein the heating unit 1 is configured On the outside of the sample chamber unit 2, for heating the sample in the sample chamber unit 2; the sample chamber unit 2 is loaded with micro-nano particles, and the sample is heated in the heating unit 1 After the chamber unit 2 is heated, a thermophoretic effect is generated in the sample chamber unit 2 to concentrate the micro-nano particles in a side of the sample chamber unit 2 away from the heating unit 1; the signal acquisition unit 4.
  • thermophoretic effect that is, the directional migration of an object under the action of a temperature gradient, and a temperature gradient field is locally generated in the sample by infrared laser irradiation, so that the exosomes in the sample migrate to a lower temperature.
  • thermophoretic effect and convection are introduced to cause a temperature difference between the micro-nano particle fluid in the sample chamber unit 2 and one side of the sample chamber unit 2, And the temperature of one side of the sample chamber unit 2 is lower than the temperature of the micro-nano particle fluid, and the thermophoretic effect causes the micro-nano particles in the sample to migrate and accumulate to the low temperature side of the sample chamber unit 2; meanwhile, since the sample fluid is subjected to Thermal expansion creates buoyancy to create convection in the sample chamber unit 2.
  • the direction of convection is directed from the periphery to the heating region of the sample chamber unit 2, as indicated by the arrow in Fig. 1, acting as a conveyor belt to concentrate the surrounding micro-nano particles in the sample chamber unit 2 On the low temperature side, it acts to accumulate micro-nano particles.
  • the heating unit 1 of the present embodiment is a laser disposed outside the sample chamber unit 2 to illuminate the inside of the sample chamber unit 2 to generate a circular heating region therein, of course, the heating region is also It can be linear or otherwise. It can be understood by those skilled in the art that the heating mode is not limited to laser irradiation, and the laser irradiation direction only needs to ensure the generation of a heat source. The choice of power depends on the direction of illumination, the diameter of the spot, the wavelength, etc., and can be based on actual conditions. Micro-nano particles and the use environment change.
  • the sample chamber unit 2 includes a sample chamber 24 loaded with a sample of micro-nanoparticles and used to provide a space for generating a thermophoresis effect, the sample chamber 24 including a first heat-conducting surface 21 for closing the sample chamber 24, and for closing The second heat conducting surface 22 of the sample chamber 24, in the present embodiment, a temperature difference between the temperature of the sample chamber 24 loaded with the micro/nanoparticle fluid and the second heat conducting surface 22 to generate a thermophoretic effect, driving the micro/nano particles from The micro-nano particle fluid is oriented to move toward the second heat transfer surface 22. Therefore, in this embodiment, the temperature in the vicinity of the second heat transfer surface 22 is lower than the temperature of the micro/nano particle fluid.
  • the sample chamber 24 is heated by a laser, the first heat conducting surface 21 and the second heat conducting surface 22 are oppositely disposed, and the second heat conducting surface 22 has a thermal conductivity greater than that of the first heat conducting surface 21, and two The heat conducting surfaces are all transparent materials for facilitating observation of the micro-nano particles.
  • the heat dissipation performance of the second heat conducting surface 22 is greater than that of the first heat conducting surface 21, and therefore, the temperature of the second heat conducting surface 22 is lower than the temperature of the first heat conducting surface 21.
  • the sample chamber 24 further includes a gasket 23 for sealing the sample chamber 24. It will be understood by those skilled in the art that the two heat conducting surfaces 21 can be disposed opposite or adjacent to each other or arranged at a predetermined angle to each other.
  • the fluid of the present embodiment may be a liquid, such as water, or a mixture of water, or a gas, such as a heated gas or a natural gas, which can be loaded with micro-nano particles and can be allowed.
  • the micro-nano particles can move freely in the fluid.
  • the first heat conducting surface 21 and the second heat conducting surface 22 are transparent, and can sequentially pass through the first heat conducting surface and the second heat conducting surface by infrared rays, and bring heat into the fluid.
  • the first heat transfer surface 21 is glass, polymethyl methacrylate (PMMA), polydimethylsiloxane (PDMS), sapphire, etc.
  • the second heat transfer surface 22 is a sapphire having good thermal conductivity. Or diamonds.
  • the laser light generates a low temperature region on the second heat transfer surface 22 by sequentially irradiating the first heat transfer surface 21, the sample chamber 24 carrying the micro/nano particles, and the second heat transfer surface 22.
  • the laser focus is adjusted into the sample chamber 24, and the sample liquid in the sample chamber 24 absorbs the laser light and the temperature rises, and the thermophoresis effect causes the micro-nano particles in the sample to migrate to the second heat-conducting surface 22 having a lower temperature, and
  • the sample liquid is thermally expanded to generate buoyancy to generate convection in the sample chamber; in the low temperature direction near the second heat transfer surface 22, the direction of convection is directed from the periphery to the laser irradiation point, and the conveyor belt acts to concentrate the surrounding micro-nano particles under the laser irradiation spot.
  • the area of the second heat transfer surface 22 of the sample chamber enhances the accumulation of micro-nano particles.
  • the micro-nano particles are selected as exosomes, and the exosomes are membranes secreted by cells, which are used for intercellular communication, and have recently become a kind of protein and genetic material related to the mother cells.
  • v T is the hot swimming velocity
  • S T is the Soret coefficient
  • D is the diffusion coefficient
  • ⁇ T is the temperature gradient
  • the negative sign at the right end of the model formula indicates that the hot swimming direction is the low temperature direction.
  • thermophoresis force of the exosomes is proportional to the square of the diameter.
  • V p is the velocity of exosomes under thermal convection
  • a is the diameter of the exosomes
  • u is the heat convection velocity
  • C D is the viscosity coefficient, which can be calculated according to formula (4), where a 1 , a 2 a 3 is a constant
  • Re s is the relative motion Reynolds number, which can be calculated according to formula (5)
  • g is the gravity acceleration
  • ⁇ p is the average density of the exosomes
  • ⁇ is the liquid density of the sample
  • ⁇ is the liquid dynamic viscosity of the sample.
  • thermophoretic force and thermal convection viscous resistance Comparing the thermophoretic force and thermal convection viscous resistance, it can be seen that the larger the object, the more dominant the thermophoretic force is, and the more likely it is to accumulate on the bottom surface of the sample chamber; the smaller the object, the more dominant the thermal convection resistance is, and the more inclined to follow Hot convection without gathering.
  • the signal amplifying unit 3 includes a microscope disposed in the micro-nanoparticle accumulation region of the sample chamber unit 2, including an objective lens 31, a mirror 32, and an observation source aligned with the accumulated micro-nano particles. 33, the micro-nano particles can be observed more clearly through the microscope.
  • the signal acquisition unit 4 is a CCD camera. Of course, it can also be any instrument capable of detecting an optical signal, and the micro-nano particles are photographed through a microscope to obtain information.
  • the exosome sample is first incubated with the fluorescently labeled aptamer to specifically bind the fluorescently labeled aptamer to the target protein on the surface of the exosomes, thereby excreting Fluorescence on the body label; the cultured exosomal sample is placed in the sample chamber 24, and the thermophoretic effect and convection are introduced by laser heating to amplify the fluorescent signal marked on the exosomes in the sample chamber; before and after the laser irradiation is recorded by the CCD Fluorescence signal, by analyzing the fluorescence signal before and after laser irradiation, to obtain the abundance of the target protein on the exosome surface; using a series of aptamers that can bind different target proteins, the surface protein map of the exosomes can be obtained, and finally through the analysis Determine the corresponding indicator parameters of the exosomes.
  • the method for detecting micro-nano particles includes:
  • Step a heating the micro-nanoparticle sample in the sample chamber unit 2 from one side, and generating a thermophoretic effect in the sample chamber unit 2 to concentrate the micro-nano particles in the low temperature one in the sample chamber unit 2 side;
  • step b the corresponding index information of the micro-nano particles is collected by analyzing the micro-nano particles accumulated on the low temperature side in the sample chamber unit 2, and the corresponding indexes are analyzed.
  • the sample fluid is thermally expanded to generate buoyancy to generate convection in the sample chamber unit 2, and in the low temperature region of the sample chamber unit 2, the direction of the convection is directed from the surroundings to the heating region of the sample chamber unit 2, and the surroundings are The micro-nano particles converge on the low temperature side of the sample chamber unit 2.
  • the present embodiment performs signal detection on the exosomes, and as shown in FIG. 2, the process is:
  • step a1 the exosome sample is incubated with the fluorescently labeled aptamer to specifically bind the fluorescently labeled aptamer to the target protein on the surface of the exosomes, thereby labeling the exosomes with fluorescence;
  • step a2 the incubated exosomal sample is placed in the sample chamber, and the thermophoretic effect and convection are introduced by laser heating, and the exosomes are concentrated on the low temperature side of the sample chamber to exosome in the sample chamber.
  • the fluorescent signal on the mark is amplified;
  • Step a3 obtaining fluorescence signals before and after light irradiation, and analyzing the fluorescence signals before and after laser irradiation to obtain abundance of target proteins on the surface of exosomes;
  • step a4 a series of exosome surface protein maps are obtained using a series of aptamers that bind to different target proteins.
  • the exosomal sample is incubated with a fluorescently labeled aptamer, and the selected aptamer is an oligonucleotide that can be specifically bound to a protein or other small molecule by an in vitro screening technique SELEX (Exponential Enrichment Ligand System Evolution).
  • Fragments, in particular, fluorescently labeled aptamers are single-stranded DNA of 20-60 bases, with a clathon diameter of less than 5 nm in the sample liquid, and exosomes having a diameter of 30-150 nm; specific recognition of CD63
  • the aptamer of the protein was applied to the exosomes in the culture supernatant of A375 cells (human melanoma cells).
  • the fluorophore can be modified at the end of the aptamer by standard means.
  • the exosomes are labeled with fluorescence carried by the aptamer.
  • the exosomal sample of the present example is a cell culture supernatant, and the incubation conditions of the sample are: 2 hours incubation time, aptamer concentration 0.1 micromoles per liter, incubation temperature room temperature.
  • the laser uses an infrared laser with a wavelength of 1480 nm for sample heating, the power is 200 mW, and the focus of the laser spot is about 200 ⁇ m. Since the main component of the sample liquid is water, the water has an absorption peak near the 1480 nm band. It is understood by those skilled in the art that the heating method is not limited to laser irradiation, and the wavelength is not limited to 1480 nm, and the laser irradiation direction is not limited to the upper direction. Under the illumination, the choice of power depends on the direction of illumination, the diameter of the spot, the wavelength, etc., and is not limited to 200 mW.
  • the laser is irradiated from top to bottom, and the upper heat conducting surface of the sample chamber is made of a transparent material such as glass, PMMA, PDMS, the lower heat conducting surface is made of sapphire having better thermal conductivity, and the low temperature area is formed on the bottom surface to make the exosomes.
  • the thermophores converge on the underside.
  • the upper heat transfer surface has a thickness of 1 mm, the lower heat transfer surface has a thickness of 1 mm, and the intermediate spacer and the sample chamber have a height of 240 mm.
  • the fluorescent label on the aptamer follows the exosomes concentrated in the bottom portion of the sample chamber below the laser spot. And produce enhanced fluorescent signal; when the aptamer does not recognize the exosome surface protein, the free aptamer cannot aggregate due to small size, and the signal is not enhanced.
  • CD63 protein is widely present on the surface of exosomes of various types of cells, and after laser irradiation, a clear fluorescent signal appears, indicating that the exosome surface of A375 cells has CD63 protein.
  • Fluorescence microscopy is used to excite and receive the fluorescent signal labeled on the aptamer after binding to the exosomes.
  • the wavelength of the excited and received fluorescence is related to the characteristics of the labeled fluorescent luminescent group.
  • the luminescent group Cy5 excitation/emission wavelength At 649/666 nm
  • the fluorescent signal was recorded by a CCD connected to a fluorescence microscope.
  • the fluorescence signal before and after laser irradiation was recorded by CCD, and the fluorescence signal before and after laser irradiation was analyzed to obtain the abundance of the target protein on the exosome surface.
  • serum samples of cervical cancer patients are used, and abundance of seven surface proteins (CD63, PTK7, EpCAM, HepG2, HER2, PSA, CA125) of exosomes in serum samples is detected by using seven different aptamers, and Compare with healthy human serum samples.
  • seven surface proteins CD63, PTK7, EpCAM, HepG2, HER2, PSA, CA125
  • exosome manipulation method used as well as the laser, sample chamber, and microscope and CCD camera are the same.
  • the serum exosomes of cervical cancer patients express high CD63 protein, and cancer-related markers PTK7, EpCAM, HepG2, HER2, PSA and CA125, among which CA125 can be used as a marker for traditional cervical cancer, and also has a part.
  • Patients with cervical cancer have high expression of HER2.
  • the tumor markers PTK7 and EpCAM are related to various cancers, HepG2 is mainly specific for liver cancer, and PSA is mainly specific for prostate cancer. However, these tumor markers are not strictly related to certain cancers.
  • CD63 is a protein that is ubiquitously expressed in exosomes, and the expression of exosomes in cancer patients is higher than that in healthy people, which is consistent with the results obtained by traditional methods.
  • the method was further applied to a large number of real clinical serum samples, including 3 cases of cervical cancer, 2 cases of ovarian cancer, 2 cases of lymphoma, 2 cases of breast cancer and 2 healthy people.
  • the method can detect the difference in the expression levels of various types of proteins of serum exosomes of various cancer patients and healthy persons.
  • the difference in serum exocytic protein expression between different types of cancer is mainly reflected in the higher expression of HER2 in breast cancer and cervical cancer, the higher expression of CA125 in ovarian cancer and cervical cancer, and the PSA in the detected cancer. None of the species were expressed, and EpCAM and PTK7 and CD63 were highly expressed in various cancers. These results are consistent with the results of the existing methods.
  • exosome surface proteins including cancer markers
  • serum of cancer patients and healthy human serum can sensitively detect the difference in the expression level of exosome surface proteins, including cancer markers, in serum of cancer patients and healthy human serum. It also shows that exosomes are more convenient, sensitive and effective as a marker for cancer tumors: traditional cancer screening or physical examination has limited types of tumor markers (limited by available expensive antibodies and reagents) and is not sensitive enough. This results in a false negative, that is, the patient does not detect the marker.
  • the CA125 expression in the venous blood test report of the cervical cancer patient in the present embodiment is within the normal range.
  • the method does not require an expensive antibody, and an aptamer capable of specifically binding to the protein of the corresponding tumor marker can be used depending on the detection.
  • the micro-nano particles are non-biological micro-nano particles, specifically fluorescent polystyrene microspheres, branded as Thermofisher, having a diameter of 50 to 200 nm, a mass fraction of 0.001%, and soluble in 0.02% of Tween20.
  • Thermofisher having a diameter of 50 to 200 nm, a mass fraction of 0.001%, and soluble in 0.02% of Tween20.
  • Tween20 aqueous solution.
  • the laser, the sample chamber, and the microscope and CCD camera were the same as in the above Examples 1 and 2.
  • the micro-nano particles are biological macromolecules such as free proteins and nucleic acids, or biological macromolecules such as proteins and nucleic acids whose exosomes are not exposed on the surface, and the thermophoretic effects of the above embodiments cannot directly accumulate free biological macromolecules. Therefore, the mechanism of the present embodiment consists in modifying an antibody or an aptamer capable of specifically binding to a target protein or a nucleic acid on a surface of a micron-sized sphere, obtaining an immunoglobulin, and incubating it with a sample containing the target biological macromolecule and The target biomacromolecules bind and label the fluorescence.
  • the microspheres are highly concentrated by the above-described thermophoresis, so that the target biomacromolecule fluorescence signal is highly amplified, and its abundance is detected by the fluorescence intensity.
  • an immunoglobulin is prepared, and the microsphere is incubated with an antibody or an aptamer to immobilize the antibody or aptamer on the surface of the microsphere to obtain an immunomicrosphere; in the process, an extra antibody that does not bind to the microsphere is added. Or the aptamer is washed away; in this embodiment, the microspheres are made of polystyrene microspheres.
  • Step b11 incubating the immunoglobulin with the sample to be detected, and the target protein or nucleic acid in the sample to be detected is specifically bound to the antibody or aptamer on the immunomicrosphere, thereby being immobilized on the immunomicrosphere;
  • Step c11 combining the immunoglobulin bound to the target biomolecule prepared in the above step b11 with the antibody or aptamer carrying the fluorophore, and labeling the target biomolecule on the immunoglobulin by specific recognition;
  • Step d11 heating the immunoglobulin sample bound to the target biomolecule in the sample chamber unit 2 from one side, and generating a thermophoretic effect in the sample chamber unit 2 to aggregate the immunomicrospheres combined with the target biomolecule On the low temperature side of the sample chamber unit 2, and amplifying the signal due to fluorescence label enrichment; in this process, by generating thermophoresis, the equivalent bio-sized molecules are captured by the immuno-microspheres to increase the equivalent size , is highly enriched and signal amplified, and the non-target biomolecule is in a free state. The equivalent size is small and the signal cannot be amplified.
  • step e11 the corresponding micro-spheres of the immunoglobulins combined with the target biomolecules are collected by the immunoglobulins of the target biomolecules accumulated on the low temperature side of the sample chamber unit 2, and the corresponding indicators are analyzed.
  • the fluorescence signal before and after the light irradiation is obtained, and the fluorescence signal of the surface of the exosomes is obtained by analyzing the fluorescence signals before and after the laser irradiation; the surface of the exosomes is obtained by using a series of aptamers capable of binding different target proteins. Protein map.
  • the immunoglobulin coated with the antibody is used to capture the free protein marker in the whole blood of ovarian cancer patients, and the infrared fluorescent laser is used to generate the thermophoresis to amplify the fluorescent signal of the protein marker, and the abundance of the protein marker to be tested is determined.
  • the results are consistent with the results of traditional assays and provide molecular information for cancer detection.
  • EpCAM, CA-125, CA19-9, CD24, HER2, MUC18, EGFR, CLDN3, CD45, CD41, D2-40 are selected as protein markers, and these protein markers are corresponding.
  • the diameter of the microspheres is not limited to 1 micrometer, as long as the size reaches the thermophoresis to converge; the material is not limited to polystyrene, as long as the material can be successfully attached to the antibody and does not affect the activity of the antibody and the protein marker to be tested, the antibody can be used.
  • the concentration and incubation temperature time are not limited to the specific values described in this example, and are varied with reference to actual use of antibody brand batches and specific experimental conditions.
  • 11 kinds of immunomicrospheres were prepared by the above steps to separately detect the above 11 kinds of markers, and the patient serum was diluted 1.1 times with 100 ⁇ L and then divided into 11 portions, and mixed with 11 kinds of immune microspheres at room temperature. After incubation for 1 hour, the fluorescently labeled antibody was incubated with the microspheres capturing the protein marker to be tested, and the protein marker was fluorescently labeled.
  • the detection system of each of the above embodiments is used for detection. The above procedure was repeated for 10 ovarian cancer patients and 10 healthy individuals, and the expression levels of 11 protein markers in 20 serum samples were measured, as shown in Figs. 7 and 8.
  • each protein marker is used as a single cancer detection standard with low accuracy. Using the sum of protein expression levels in 11 as a test standard, the test can accurately distinguish between ovarian cancer and healthy samples.
  • the use of more diagnostic markers will greatly improve the diagnostic accuracy, but the cost increases with the number of markers, especially antibodies against certain markers are rare and expensive.
  • each marker requires only 1 ng of antibody per person, and the cost is less than 1 yuan, and no other expensive reagent is needed.

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Abstract

一种微纳粒子检测系统及方法，所述系统包括加热单元（1）、样品仓室单元（2）、信号采集单元（4），其中，所述加热单元（1）设置在所述样品仓室单元（2）的外侧，用以对所述样品仓室单元（2）内的样品加热；所述样品仓室单元（2）内装载有微纳粒子流体，在所述加热单元（1）对所述样品仓室单元（2）加热后，所述样品仓室单元（2）内产生热泳效应，以将微纳粒子汇聚在所述样品仓室单元（2）内温度低于微纳粒子流体的一侧；所述信号采集单元（4）采集汇聚的所述微纳粒子的相关信息，并进行相应分析。微纳检测系统通过采用热泳效应对粒子进行聚积，只需微量的微纳粒子即可完成汇聚及检测，并且无需样品前处理和微纳粒子提纯，通用于适体和抗体，具有广阔的应用前景。

Description

微纳粒子检测系统及方法 技术领域
本发明涉及微纳粒子检测技术领域，尤其涉及一种基于热泳效应的微纳粒子检测系统及方法。
背景技术
现有技术中对微纳粒子进行检测，以测量微粒大小、形状、浓度、活性等，在血液学、免疫学、分子生物学、临床医学等学科有较为广泛的应用。现有技术中常采用流式微粒检测方法对微纳粒子进行检测，其是对处于液体中的微粒颗粒逐个进行定量分析和分选的技术，在检测中所采用的库尔特原理是指：悬浮在电解液中的颗粒随电解液通过小孔时，取代相同体积的电解液，在恒电流设计的电路中导致小孔内外两电极间电阻发生瞬间变化，产生电位脉冲，脉冲信号的大小和次数与颗粒的大小和数目成正比。样品聚焦是流式微粒检测的关键技术，目前的检测中都是通过外力作用对样品液实现聚焦。聚焦又分为通过鞘液聚焦和无需鞘液的聚焦。
其中，鞘液聚焦如中国专利201210482142.7公开的《微流体微粒仪及制作方法》中，利用外界注射泵的压力分别从样品液入口注入样品液，从鞘液入口注入鞘液，然后样品液和两路鞘液同时流到鞘流汇聚区，鞘液的聚集作用将样品液中的微粒颗粒包夹成线性排列流入检测区进行检测。这种方法中两个鞘流和样品液都需要驱动源，采用一个电机控制三个管道的方式，这样不仅设备变得很庞大，成本也提高，更为重要的是由于每次进行检测时需要更换芯片，那么每次都检测都需要重新将三个通道与电机进行连接，这个连接处的密封性问题就会影响到对三个通道的压力的大小，造成聚焦效果不好，测试结果就不够 精确。
其中，无需鞘液的聚焦，如中国专利201310283051.5公开的《一种用于流式微粒仪的微流控芯片结构及其制作方法》，其采用锥形聚焦结构，认为其具有类似与传统的鞘液流系统的聚焦效果，使得微粒颗粒单个流入微通道，微通道通过通道束缚微粒使其单个通过检测区，在高浓度样本的检测条件下造成检测结果的不精确。
在上述两种检测微纳粒子的技术方案中，一方面，通过产生电位脉冲，采用电化学方法对纳米颗粒进行分离及检测，形成含微纳粒子的流束，所需样品的量极大；另一方面，通过诸如电机的驱动源，同时采用固定结构的单一的通道，限定微纳粒子的流动方向及聚积方向，在施加外作用力以及通道限定的过程中，外力作用于流体，往往针对于微纳粒子的施力是不可控的。
尤其针对微纳生物粒子，诸如外泌体的检测，外泌体是由细胞分泌的膜泡，用于细胞间交流，因其含有与母细胞相关的蛋白及遗传物质，可调节多种生理或病理反应，包括肿瘤细胞侵袭与转移、血管生长、免疫应答等，近年来逐渐成为一种新兴的非侵入式肿瘤诊断的生物标志物。外泌体用于肿瘤诊断常需要分析其表面蛋白类型，但由于缺乏准确可行且易操作的分析方法，使得目前在分析不同外泌体表面蛋白质的微小差别上仍面临挑战。
现有技术中通常采用：其一，酶联免疫吸附测定，即ELISA，指将可溶性的抗体结合到聚苯乙烯等固相载体上，利用抗原抗体结合特异性进行免疫反应的定性和定量检测方法，在测定时，把受检标本(测定其中的抗体)和酶标抗体按不同的步骤与固相载体表面的抗原起反应；用洗涤的方法使固相载体上形成的抗原抗体复合物与其他物质分开，最后结合在固相载体上的酶量与标本中受检物质的量成一定的比例。加入酶反应的底物后，底物被酶催化变为有色产物， 产物的量与标本中受检物质的量直接相关，故可根据颜色反应的深浅刊物定性或定量分析。
其二，蛋白质印迹法，即Western Blot，其基本原理是通过特异性抗体对凝胶电泳处理过的细胞或生物组织样品进行着色；通过分析着色的位置和着色深度获得特定蛋白质在所分析的细胞或组织中表达情况的信息。
上述两种技术方案，一方面对样本进行复杂前处理、分离及提纯、繁重操作步骤，需采用特殊的设备及方法；另一方面，检测方法需要采用大量的样品，往往针对血清进行癌变检测的过程不具有适应性。
发明内容
本发明的目的在于提供一种微纳粒子检测系统及方法，用以克服上述技术缺陷。
为实现上述目的，本发明提供一种微纳粒子检测系统，包括加热单元、样品仓室单元，其中，
所述加热单元，用以向所述样品仓室单元内的样品加热；
所述样品仓室单元内装载有微纳粒子流体，在所述加热单元对所述样品仓室单元加热后，所述样品仓室单元内产生热泳效应，以将微纳粒子汇聚在所述样品仓室单元内温度低于微纳粒子流体的一侧，用以检测。
进一步地，所述系统还包括信号采集单元，所述信号采集单元采集汇聚的所述微纳粒子的相关信息，并进行相应分析。
进一步地，所述样品仓室单元包括装载所述微纳粒子流体并用以提供产生热泳效应空间的密闭样品室，所述样品室包括：用以封闭所述样品室并聚积所述微纳粒子的第二导热面，所述第二导热面附近温度低于所述微纳粒子流体的 温度，以在所述第二导热面与微纳粒子流体之间产生温差，产生热泳效应，驱使微纳粒子向第二导热面定向移动。
进一步地，所述加热单元为激光器，其向所述样品仓室单元照射，光束依次通过所述微纳粒子流体和第二导热面，以对所述微纳粒子溶液产生热泳效应。
进一步地，所述样品室还包括：用以封闭所述样品室的第一导热面，所述第二导热面和第一导热面均可使光束通过。
进一步地，所述第二导热面为透明材质，其为蓝宝石材质；所述第一导热面为玻璃、聚甲基丙烯酸甲酯、聚二甲基硅氧烷、蓝宝石、钻石中的任一种或任几种的组合。
进一步地，所述微纳粒子为外泌体、细胞外囊泡、细胞或生物相容性良好的微球。
进一步地，所述微纳粒子为结合有目标生物分子的免疫微球，免疫微球为抗体或适体固定在微球表面而制得。
本发明还提供一种微纳粒子检测方法，其特征在于，包括：对样品仓室单元内的荧光标记的微纳粒子流体进行加热，在样品仓室单元内产生温差，以在样品仓室单产生热泳效应，以将荧光标记的微纳粒子汇聚在所述样品仓室单元温度低于微纳粒子流体的一侧，以将标记的荧光信号放大；
步骤b，通过对所述样品仓室单元内的低温一侧聚积的微纳粒子，采集微纳粒子的相应指标信息并对相应指标进行分析。
进一步地，所述微纳粒子为外泌体或结合有目标生物分子的免疫微球，免疫微球为抗体或适体固定在微球表面而制得。
与现有技术相比本发明的有益效果在于，本发明微纳检测系统通过对微纳粒子所在的样品仓室单元的其中一方向进行加热，引入热泳效应及对流，使样 品仓室单元产生温差，在远离加热单元的一侧产生低温，热泳效应使样品中微纳粒子迁移并聚积到样品仓室单元，以完成微纳粒子的聚积；同时，由于样品液体受热膨胀产生浮力从而在样品仓室单元中产生对流，在样品仓室单元的低温区域，对流的方向从周围指向样品仓室单元的加热区域，进一步促进微纳粒子的聚积。样品室的下表面设计为导热性极好的透明材料，致使外泌体向温度较低的样品室下表面迁移。同时由于样品液体受热膨胀产生浮力从而在样品室中产生对流，对流能够加速和加强外泌体的汇聚，从而提高信号放大倍率。进一步地，本系统将含有外泌体的待测样品与荧光标记的适体或抗体孵育，通过适体或抗体与外泌体表面蛋白特异性结合将外泌体标记上荧光；将孵育好的样品放入上下透明的样品室，并放置在荧光显微镜载物台上进行观测，红外激光器照射透过样品室作用于样品，通过热泳作用将样品中外泌体高度富集在样品室底部的激光光点处，使得外泌体荧光高度放大，并通过荧光强弱检测某种外泌体表面蛋白的丰度。
进一步地，本系统采用激光对样品室照射进行加热，通过在样品室相对侧面设置导热性能不同的透明导热面，在两个导热面之间产生温差，以产生热泳效应，驱使微纳粒子从第一导热面低温较低的第二导热面定向移动。尤其采用光束加热，不需要采用其他辅助设备，只需样品室上下设置透明的导热面即可。并且，微纳粒子在热泳效应下受力与粒子直径平方成正比，而与微纳粒子数量多少无关，因此，只需微量的微纳粒子即可完成汇聚及检测，对于外泌体仅需0.1微升的样本用量，操作方便，无需特殊仪器，并且无需样品前处理和外泌体提纯，通用于适体和抗体；不限于外泌体，其他细胞外囊泡、细胞等微纳生物粒子均可。
尤其是，本发明微纳粒子检测系统及方法，可以选择特定温度下完成 测量，不受具体温度的限定，只需能够产生温差以聚积粒子即可；还可在各种不同的溶液环境中完成测量，包括研究膜蛋白所需的复杂的去污剂环境；还可对各种不同大小的分子：如离子、核酸片段、核小体、脂质体进行检测，在具体进行检测时，系统可根据颗粒自身的物理性能及颗粒的大小，调整温差、上下导热面之间的高度、流体的种类及激光照射的频率等参数进行调整，上述各个参量的调整，均能够实现定量的调整，控制精准，调节方便。
本发明游离蛋白、核酸等生物大分子或者外泌体中未暴露在表面的蛋白、核酸等生物大分子，在微米尺寸的球体表面修饰能与目标蛋白、核酸特异性结合的抗体或适体，得到免疫微球，并将其与含有目标生物大分子的样品孵育并与目标生物大分子结合并标记荧光，以对游离态的粒子或未暴露在表面的目标生物大分子进行汇聚，并在汇聚后进行检测。
本发明基于热泳效应聚积粒子，对微纳粒子的装载容器没有限定，尤其在体积较大的容器中，粒子更容易在热泳效应下聚积，而不需考虑采用毛细管等载体容器进行引导。
附图说明
图1为本发明的微纳粒子检测系统的结构框图；
图2为本发明的基于外泌体的信号检测流程框图；
图3为本发明实施例1的外泌体在试验前后的对比图谱；
图4为本发明实施例2的外泌体在检测后的各表面蛋白图谱及对应的各表面蛋白表达量示意图；
图5为本发明实施例2各类癌症患者于健康人的血清外泌体的各类蛋白表达量的示意图；
图6为本发明实施例3的不同直径的荧光聚苯乙烯微球的荧光测量灰度值示意图；
图7为本发明实施例4的卵巢癌患者和健康人血清中11种蛋白标志物表达量的示意图；
图8为本发明实施例4的11不同标志物和其总和作为区别癌症与健康标准的正确率示意图。
具体实施方式
以下结合附图，对本发明上述的和另外的技术特征和优点作更详细的说明。
下面参照附图来描述本发明的优选实施方式。本领域技术人员应当理解的是，这些实施方式仅仅用于解释本发明的技术原理，并非旨在限制本发明的保护范围。
需要说明的是，在本发明的描述中，术语“上”、“下”、“左”、“右”、“内”、“外”等指示的方向或位置关系的术语是基于附图所示的方向或位置关系，这仅仅是为了便于描述，而不是指示或暗示所述装置或元件必须具有特定的方位、以特定的方位构造和操作，因此不能理解为对本发明的限制。此外，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性。
此外，还需要说明的是，在本发明的描述中，除非另有明确的规定和限定，术语“安装”、“相连”、“连接”应做广义理解，例如，可以是固定连接，也可以是可拆卸连接，或一体地连接；可以是机械连接，也可以是电连接；可以是直接相连，也可以通过中间媒介间接相连，可以是两个元件内部的连通。对于本领域技术人员而言，可根据具体情况理解上述术语在本发明中的具体含义。
请参阅图1所示，其为本发明的微纳粒子检测系统的结构框图，本实施例的系统包括加热单元1、样品仓室单元2、信号采集单元4，其中，所述加热单元1设置在所述样品仓室单元2的外侧，用以向所述样品仓室单元2内的样品加热；所述样品仓室单元2内装载有微纳粒子，在所述加热单元1对所述样品仓室单元2加热后，所述样品仓室单元2内产生热泳效应，以将微纳粒子汇聚在所述样品仓室单元2内远离所述加热单元1的一侧；所述信号采集单元4，其在所述样品仓室单元2内的微纳粒子聚积后，采集所述微纳粒子的相关信号信息，并对相应的微纳粒子进行相应分析。本系统利用热泳效应，即物体在温度梯度作用下的定向迁移，通过红外激光照射在样品局部产生温度梯度场，使样品中的外泌体迁移到温度较低处。通过对微纳粒子所在的样品仓室单元2的其中一方向进行加热，引入热泳效应及对流，使样品仓室单元2中的微纳粒子流体与样品仓室单元2的一侧产生温差，并使样品仓室单元2的一侧的温度低于微纳粒子流体的温度，热泳效应使样品中微纳粒子迁移并聚积到样品仓室单元2的低温一侧；同时，由于样品流体受热膨胀产生浮力从而在样品仓室单元2中产生对流。在样品仓室单元2的低温区域，对流的方向从周围指向样品仓室单元2的加热区域，如图1中箭头所指方向，起到传送带作用将周围微纳粒子汇聚在样品仓室单元2的低温一侧，从而起到聚积微纳粒子的作用。
具体而言，本实施例的加热单元1为激光器，其设置在样品仓室单元2的外侧，对样品仓室单元2内进行照射，以在其内部产生圆形的加热区域，当然加热区域也可以为线性或者其它方式。本领域技术人员可以理解的是，加热方式并不仅限于激光照射，激光照射方向只需能够确保产生热源即可，功率的选择取决于照射方向，光点直径、波长等因素，均可根据实际的微纳粒子及使用环境而改变。
具体而言，样品仓室单元2包括装载微纳粒子样品并用以提供产生热泳效应空间的密闭样品室24，样品室24包括用以封闭样品室24的第一导热面21，以及用以封闭样品室24的第二导热面22，在本实施例中，样品室24的装载有微纳粒子流体的温度与第二导热面22之间产生温差，以产生热泳效应，驱使微纳粒子从微纳粒子流体向第二导热面22定向移动。因此，本实施例将所述第二导热面22的附近的温度低于微纳粒子流体的温度。
在本实施例中，采用激光器对样品室24进行加热，所述第一导热面21、第二导热面22相对设置，所述第二导热面22的导热性大于第一导热面21，且两个导热面均为透明材质，便于对微纳粒子进行观测第二导热面22的散热性能大于第一导热面21，因此，第二导热面22的温度低于第一导热面21的温度。所述样品室24还包括用以密封样品室24的垫片23，本领域技术人员可以理解的是，两个导热面21可相对设置或相邻设置，或相互之间按照预设夹角布置，只需驱使微纳粒子沿设定的某一方向运动、聚积即可。本领域技术人员可以理解的是，本实施例的流体可以为液体，如水，或水的混合液，也可以为气体，如加热气体或自然态气体，只需能够装载微纳粒子，并能够允许微纳粒子在流体中自由移动即可。同时，第一导热面21和第二导热面22为透明的，可以通过红外线依次穿过第一导热面和第二导热面，并将热量带入所述流体中。
作为较优的实施例，第一导热面21为玻璃、聚甲基丙烯酸甲酯(PMMA)、聚二甲基硅氧烷(PDMS)、蓝宝石等，第二导热面22为导热性良好的蓝宝石或钻石。激光通过顺次照射第一导热面21、装载微纳粒子的样品室24、第二导热面22，在第二导热面22产生低温区。将激光焦点调节至样品室24内，样品室24内激光经过区域的样品液体吸收激光并温度升高,热泳效应使样品中微纳粒子迁移到温度较低的第二导热面22，同时由于样品液体受热膨胀产生浮力从而 在样品室中产生对流；在第二导热面22附近的低温方向，对流的方向从周围指向激光照射点，起到传送带作用将周围微纳粒子汇聚在激光照射点下方的样品室第二导热面22的区域，从而增强微纳粒子聚积。
本实施例中，微纳粒子选择为外泌体，外泌体是由细胞分泌的膜泡，用于细胞间交流，因其含有与母细胞相关的蛋白及遗传物质，近年来逐渐成为一种新兴的非侵入式肿瘤诊断的生物标志物。
本实施例基于外泌体的具体原理如下所述。
外泌体热泳运动模型：
v T＝-S TD▽T  (1)
其中v T为热泳速度，S T为Soret系数，D为扩散系数，▽T为温度梯度，模型公式右端负号表示热泳方向为低温方向。
上述公式(1)中的Soret系数计算公式：
Figure PCTCN2018098650-appb-000001
其中A为外泌体表面积，k为玻尔兹曼常数，T为温度，s hyd为水合作用熵，β为系数，σ eff为外泌体表面等效电荷密度，λ DH为德拜长度，ε 0为真空介电常数，ε相对介电常数。综合上述公式(1)-(2)，可知外泌体热泳受力与直径平方成正比。
外泌体在热对流中迁移模型：
Figure PCTCN2018098650-appb-000002
Figure PCTCN2018098650-appb-000003
Re s＝ρa|u-V p|/η  (5)
其中V p为外泌体在热对流作用下的运动速度，a为外泌体直径，u为热对流速度，C D为黏性系数，可根据公式(4)计算，其中a 1、a 2、a 3为常数，Re s为相对运动雷诺数，可根据公式(5)计算，g是重力加速度，ρ p为外泌体平均密度，ρ为样品液体密度，η为样本液体动力黏度。综合公式(3)-(5)，可知外泌体受热对流的黏性阻力与直径成正比。
对比热泳力与热对流黏性阻力，可知物体越大，受到的热泳力越占主导，越倾向于聚集在样品室底面；物体越小，受到热对流黏性阻力越占主导，越倾向于跟随热对流而不聚集。
继续参阅图1所示，信号放大单元3包括设置在所述样品仓室单元2的微纳粒子聚积区域的显微镜，其包括对准聚积的微纳粒子的物镜31、反光镜32、以及观测光源33，通过显微镜更能清晰的观测微纳粒子。所述信号采集单元4为CCD相机，当然，也可以是任何能够探测光信号的仪器，通过显微镜对微纳粒子进行拍照，获取信息。
在本实施例中，对外泌体信号检测而言，首先将外泌体样品与荧光标记的适体孵育，使荧光标记的适体与外泌体表面的靶蛋白特异性结合，从而将外泌体标记上荧光；将孵育后的外泌体样品放入样品室24，并通过激光加热引入热泳效应和对流，将样品室内的外泌体上标记的荧光信号放大；通过CCD记录激光照射前后的荧光信号，通过分析激光照射前后的荧光信号，得出外泌体表面靶蛋白的丰度；使用一系列能结合不同靶蛋白的适体，能够得出外泌体表面蛋 白图谱，并通过该分析最终确定外泌体的相应指标参数。
本实施例中，微纳粒子的检测方法包括：
步骤a，从一侧对样品仓室单元2内的微纳粒子样品进行加热，在样品仓室单元2产生热泳效应，以将微纳粒子汇聚在所述样品仓室单元2内的低温一侧；
步骤b，通过对所述样品仓室单元2内的低温一侧聚积的微纳粒子，采集微纳粒子的相应指标信息并对相应指标进行分析。
在上述步骤a中，样品流体受热膨胀产生浮力从而在样品仓室单元2中产生对流，在样品仓室单元2的低温区域，对流的方向从周围指向样品仓室单元2的加热区域，将周围微纳粒子汇聚在样品仓室单元2的低温一侧。
具体而言，本实施例对外泌体进行信号检测，结合图2所示，该过程为：
步骤a1，将外泌体样品与荧光标记的适体孵育，使荧光标记的适体与外泌体表面的靶蛋白特异性结合，从而将外泌体标记上荧光；
步骤a2，将孵育后的外泌体样品放入样品室，并通过激光加热引入热泳效应和对流，将外泌体汇聚在所述样品室内的低温一侧，以将样品室内的外泌体上标记的荧光信号放大；
步骤a3，获取光照射前后的荧光信号，通过分析激光照射前后的荧光信号，得出外泌体表面靶蛋白的丰度；
步骤a4，使用一系列能结合不同靶蛋白的适体，得出外泌体表面蛋白图谱。
下面通过具体实施例对上述微纳粒子检测系统和方法进行说明。
实施例1
将外泌体样品与荧光标记的适体孵育，选择的适体是经体外筛选技术SELEX(指数富集配体系统进化)筛选出的能特异结合蛋白质或其他小分子物质的寡聚核苷酸片段，具体而言，荧光标记的适体为20-60碱基的单链DNA，在样 品液体中的线团直径小于5纳米，而外泌体直径为30-150纳米；将特异性识别CD63蛋白的适体应用于A375细胞(人黑色素瘤细胞)培养基上清中的外泌体。通过标准手段可在适体端部修饰荧光基团，当适体与外泌体表面的靶蛋白之间的特异性相互作用时，外泌体标记了适体携带的荧光。本实施例的外泌体样品为细胞培养基上清，样品的孵育条件均为：2小时孵育时间、适体浓度0.1微摩尔每升、孵育温度室温。
其中，激光器采用1480nm波长的红外激光用于样品加热，功率为200毫瓦，焦点出激光光斑直径约200微米。因样品液体一般主要成分为水，水对1480nm波段附近有一个吸收峰，本领域技术人员可以理解的是，加热方式并不仅限于激光照射，波长也不仅限于1480nm，激光照射方向不限于从上向下照射，功率的选择取决于照射方向，光点直径、波长等因素，不限于200毫瓦。在本实施例中，激光从上至下照射，样品室的上导热面采用透明材质，如玻璃、PMMA、PDMS，下导热面采用导热性更好的蓝宝石，在底面形成低温区使外泌体热泳汇聚于底面。上导热面的厚度为1mm，下导热面的厚度为1mm，中间垫片以及样品室的高度均为240mm。
按照上述基于外泌体的信号检测方法进行操作，当适体识别外泌体表面蛋白并与之结合时，适体上的荧光标记跟随外泌体被汇聚于激光光点下方的样品室底部区域，并产生增强荧光信号；当适体未识别外泌体表面蛋白时，游离的适体由于尺寸小不能汇聚，信号不增强。结合图3所示，本实施例中，CD63蛋白广泛存在于各类细胞的外泌体表面，通过激光照射后，出现明显的荧光信号，表明A375细胞的外泌体表面具有CD63蛋白。
采用荧光显微镜激发和接收与外泌体结合后的适体上标记的荧光信号，激发和接收荧光的波长与标记的荧光发光基团特性相关，本实施例中，发光基团 Cy5激发/发射波长为649/666nm，荧光信号被与荧光显微镜连接的CCD记录。通过CCD记录激光照射前后的荧光信号，通过分析激光照射前后的荧光信号，得出外泌体表面靶蛋白的丰度。
实施例2
本实施例，采用宫颈癌患者血清样品，使用7种不同的适体对血清样品中外泌体7种表面蛋白(CD63、PTK7、EpCAM、HepG2、HER2、PSA、CA125)的丰度进行检测，并与健康人血清样品进行对比。
采用的外泌体操作方法，以及激光器、样品室及显微镜和CCD相机均相同。
结合图4所示，可知此宫颈癌患者血清外泌体高表达CD63蛋白，和癌症相关标志物PTK7、EpCAM、HepG2、HER2、PSA以及CA125，其中CA125可作为传统的宫颈癌的标志物，也有部分宫颈癌患者存在HER2高表达。一般认为肿瘤标志物PTK7、EpCAM与多种癌症相关，HepG2主要针对肝癌有特异性，PSA主要针对前列腺癌有特异性。但这些肿瘤标志物并不是与某种癌症具有严格的对应相关关系。但由于肿瘤在生长或者癌症向其他器官转移过程中，经过多次分裂增殖，细胞不断产生基因突变，呈现出分子生物学或基因方面的改变，所以这些肿瘤标志物并不是与某种癌症具有严格的对应相关关系。本实施例中此宫颈癌患者血清中检测出PTK7、EpCAM、HepG2，体现了此方法在捕获肿瘤的基因突变或转移的潜力。此外CD63作为外泌体普遍表达的蛋白，在癌症患者外泌体的表达也高于健康人的，与已有传统检测方法得到的结果相符。
进一步将本方法运用于大量真实临床血清样品，包含3例宫颈癌、2例卵巢癌、2例淋巴癌、2例乳腺癌以及2例健康人。结合图5所示，本方法能够检测出各类癌症患者与健康人的血清外泌体的各类蛋白表达量的区别。对不同种类癌症之间，血清外泌体蛋白表达量的区别，主要表现在HER2在乳腺癌和宫颈癌 中表达较高，CA125在卵巢癌和宫颈癌中表达较高，PSA在所检测的癌症中种类中均不表达，EpCAM和PTK7和CD63在多种癌症中具有较高表达。这些结果均与已有方法检测结果相符。
说明本方法能够灵敏地探测到癌症患者血清与健康人血清中的外泌体表面蛋白，包括癌症标志物的表达量的区别。并且表明以外泌体作为癌症肿瘤标记物的检测方法更为方便、灵敏、有效：传统癌症筛查或体检检测的肿瘤标记物种类有限(受限于可用的昂贵抗体和试剂)且灵敏度不高而导致假阴性，即患者未检测到标记物，例如，本实施例中宫颈癌患者静脉血检测报告中CA125表达结果为在正常范围值内。而本方法无需昂贵抗体，根据检测需要可使用能与相应肿瘤标志物的蛋白特异性结合的适体即可。
实施例3
本实施例，采用的微纳粒子为非生物微纳粒子，具体为荧光聚苯乙烯微球，品牌为Thermofisher，直径为50至200纳米，质量分数为0.001％，溶于含0.02％的Tween20的水溶液中。激光器、样品室及显微镜和CCD相机均与上述实施例1、2相同。
结合下图5所示，所有不同直径的荧光微球均高度汇聚于激光光点处，且根据荧光测量灰度值与荧光图片可见汇聚程度与荧光强度均随着颗粒直径增大而增强，与本实施例工作原理相符，即大颗粒更倾向于汇聚。本实施例说明无论是生物还是非生物的微纳粒子，均适用于本技术方案的构思。
实施例4
本实施例微纳粒子为游离蛋白、核酸等生物大分子或者外泌体未暴露在表面的蛋白、核酸等生物大分子，采用上述各实施例的热泳效应不能直接聚积游离态的生物大分子，因此，本实施例的机理在于，在微米尺寸的球体表面修饰 能与目标蛋白、核酸特异性结合的抗体或适体，得到免疫微球，并将其与含有目标生物大分子的样品孵育并与目标生物大分子结合并标记荧光。通过上述热泳作用将微球高度汇聚，使得目标生物大分子荧光信号高度放大，并通过荧光强弱检测其丰度。
本实施例基于微球载体的粒子检测方法包括：
步骤a11,制备免疫微球，将微球与抗体或适体共同孵育，使抗体或适体固定在微球表面,得到免疫微球；在该过程中，将多余的未与微球结合的抗体或适体洗去；在本实施例中，微球采用聚苯乙烯微球。
步骤b11，将免疫微球与待检测样品进行孵育，待检测样品中的目标蛋白或核酸与免疫微球上的抗体或适体进行特异性结合，从而固定在免疫微球上；
步骤c11，将上述步骤b11制得的结合有目标生物分子的免疫微球与携带荧光基团的抗体或适体结合，通过特异性识别，将免疫微球上的目标生物分子标记荧光；
步骤d11，从一侧对样品仓室单元2内的结合有目标生物分子的免疫微球样品进行加热，在样品仓室单元2产生热泳效应，以将结合有目标生物分子的免疫微球汇聚在所述样品仓室单元2内的低温一侧，并由于荧光标记富集而使信号放大；在该过程中，通过产生热泳，由于目标生物分子被免疫微球捕获从而等效尺寸变大，被高度富集并信号放大，而非目标生物分子处于游离状态等效尺寸很小信号无法被放大。
步骤e11，通过对所述样品仓室单元2内的低温一侧聚积的结合有目标生物分子的免疫微球，采集结合有目标生物分子的免疫微球的相应指标信息并对相应指标进行分析。在该过程中，获取光照射前后的荧光信号，通过分析激光照射前后的荧光信号，得出外泌体表面靶蛋白的丰度；使用一系列能结合不同靶 蛋白的适体，得出外泌体表面蛋白图谱。
本实例采用表面包被抗体的免疫微球捕获卵巢癌症患者全血中游离蛋白标志物，采用红外激光产生热泳对蛋白标志物荧光信号进行放大检测，并确定待测蛋白标志物的丰度，其结果与传统检测方法结果符合，为癌症检测提供分子信息。在本实施例中，针对卵巢癌，选取EpCAM、CA-125、CA19-9、CD24、HER2、MUC18、EGFR、CLDN3、CD45、CD41、D2-40作为蛋白标志物，将这些蛋白标志物对应的特异性抗体(从abcam公司购买)分别制成免疫微球，每种抗体单独制备微球，专门针对一种标志物的检测。对于包被抗体免疫微球的制备有标准流程可参考，在此简要叙述：将直径1微米聚苯乙烯微球与5μg/ml浓度的抗体在室温下孵育1小时，孵育后用超滤法除去多余未反应的抗体。在此微球的直径不限于1微米，只要尺寸达到热泳能够汇聚；材质不限于聚苯乙烯，只要能够成功将抗体附着并不影响抗体和待测蛋白标志物活性的材质均可使用，抗体浓度和孵育温度时间均不限于本实例所述具体数值，参考实际使用抗体品牌批次和具体实验条件而变化。
本实施例，采用以上步骤制备了11种免疫微球，以分别检测上述11种标志物，将取患者血清1.1μL稀释100倍后均分11份，分别与11种免疫微球混合在室温下孵育1小时，将具有荧光标记的抗体与捕获待测蛋白标志物的微球进行孵育，对蛋白标志物进行荧光标记。并采用上述各实施例的检测系统进行检测。对10例卵巢癌患者和10例健康人重复上述步骤，测得20例血清样品中11种蛋白标志物表达量，参阅图7和图8所示。由于癌症的异质性，具体每例患者血清的标志物表达不完全相同，但总体上表达量明显高于健康样本。重要的是每种蛋白标志物作为单一癌症检测标准，准确率不高。采用11中蛋白表达量之和作为检测标准，测能够准确区分卵巢癌与健康样本。采用更多的有诊断 意义的标志物将大大提高诊断准确率，但成本随标志物数量增长而上升，尤其针对某种标志物的抗体较为罕见昂贵。本实施例的检测方法，每种标志物每人仅需1ng抗体，成本不足1元，无需其他昂贵试剂。
至此，已经结合附图所示的优选实施方式描述了本发明的技术方案，但是，本领域技术人员容易理解的是，本发明的保护范围显然不局限于这些具体实施方式。在不偏离本发明的原理的前提下，本领域技术人员可以对相关技术特征作出等同的更改或替换，这些更改或替换之后的技术方案都将落入本发明的保护范围之内。

Claims (10)

  1. 一种微纳粒子检测系统，其特征在于，包括加热单元、样品仓室单元，其中，
    所述加热单元，用以向所述样品仓室单元内的样品加热；
    所述样品仓室单元内装载有微纳粒子流体，在所述加热单元对所述样品仓室单元加热后，所述样品仓室单元内产生热泳效应，以将微纳粒子汇聚在所述样品仓室单元内温度低于微纳粒子流体的一侧，用以检测。
  2. 根据权利要求1所述的微纳粒子检测系统，其特征在于，所述系统还包括信号采集单元，所述信号采集单元采集汇聚的所述微纳粒子的相关信息，并进行相应分析。
  3. 根据权利要求1所述的微纳粒子检测系统，其特征在于，所述样品仓室单元包括装载所述微纳粒子流体并用以提供产生热泳效应空间的密闭样品室，所述样品室包括：用以封闭所述样品室并聚积所述微纳粒子的第二导热面，所述第二导热面附近温度低于所述微纳粒子流体的温度，以在所述第二导热面与微纳粒子流体之间产生温差，产生热泳效应，驱使微纳粒子向第二导热面定向移动。
  4. 根据权利要求3所述的微纳粒子检测系统，其特征在于，所述加热单元为激光器，其向所述样品仓室单元照射，光束依次通过所述微纳粒子流体和第二导热面，以对所述微纳粒子溶液产生热泳效应。
  5. 根据权利要求4所述的微纳粒子检测系统，其特征在于，所述样品室还包括：用以封闭所述样品室的第一导热面，所述第二导热面和第一导热面均可使光束通过。
  6. 根据权利要求5所述的微纳粒子检测系统，其特征在于，所述第二导热面为透明材质，其为蓝宝石或钻石材质；
    所述第一导热面为玻璃、聚甲基丙烯酸甲酯、聚二甲基硅氧烷、蓝宝石中的任一种或任几种的组合。
  7. 根据权利要求1-6任一项所述的微纳粒子检测系统，其特征在于，所述微纳粒子为外泌体、细胞外囊泡、细胞或生物相容性良好的微球。
  8. 根据权利要求1-6任一项所述的微纳粒子检测系统，其特征在于，所述微纳粒子为结合有目标生物分子的免疫微球，免疫微球为抗体或适体固定在微球表面而制得。
  9. 一种微纳粒子检测方法，其特征在于，包括：
    对样品仓室单元内的荧光标记的微纳粒子流体进行加热，在样品仓室单元内产生温差，以在样品仓室单产生热泳效应，以将荧光标记的微纳粒子汇聚在所述样品仓室单元温度低于微纳粒子流体的一侧，以将标记的荧光信号放大；
    步骤b，通过对所述样品仓室单元内的低温一侧聚积的微纳粒子，采集微纳粒子的相应指标信息并对相应指标进行分析。
  10. 根据权利要求1所述的微纳粒子检测方法，其特征在于，所述微纳粒子为外泌体或结合有目标生物分子的免疫微球，免疫微球为抗体或适体固定在微球表面而制得。
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