WO2021120944A1 - 一种微纳颗粒浓度的检测方法 - Google Patents

一种微纳颗粒浓度的检测方法 Download PDF

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WO2021120944A1
WO2021120944A1 PCT/CN2020/128991 CN2020128991W WO2021120944A1 WO 2021120944 A1 WO2021120944 A1 WO 2021120944A1 CN 2020128991 W CN2020128991 W CN 2020128991W WO 2021120944 A1 WO2021120944 A1 WO 2021120944A1
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micro
particle
particles
tested
nano particles
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PCT/CN2020/128991
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English (en)
French (fr)
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熊贵
柳可
王哲
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瑞芯智造(深圳)科技有限公司
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Priority to US17/787,298 priority Critical patent/US20230015660A1/en
Priority to EP20903564.1A priority patent/EP4071457A4/en
Priority to JP2022538383A priority patent/JP2023507036A/ja
Publication of WO2021120944A1 publication Critical patent/WO2021120944A1/zh

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    • 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
    • G01N15/02—Investigating particle size or size distribution
    • G01N15/0205—Investigating particle size or size distribution by optical means
    • G01N15/0227—Investigating particle size or size distribution by optical means using imaging; using holography
    • 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
    • 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
    • G01N15/0656—Investigating concentration of particle suspensions using electric, e.g. electrostatic methods or magnetic methods
    • 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
    • G01N15/10—Investigating individual particles
    • G01N15/1031—Investigating individual particles by measuring electrical or magnetic effects
    • 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
    • G01N15/10—Investigating individual particles
    • G01N15/1031—Investigating individual particles by measuring electrical or magnetic effects
    • G01N15/12—Investigating individual particles by measuring electrical or magnetic effects by observing changes in resistance or impedance across apertures when traversed by individual particles, e.g. by using the Coulter principle
    • 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
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1429—Signal processing
    • G—PHYSICS
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    • 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
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1429—Signal processing
    • G01N15/1433—Signal processing using image recognition
    • 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
    • G01N33/54346—Nanoparticles
    • 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
    • G01N15/075—Investigating concentration of particle suspensions by optical means
    • 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
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    • 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
    • G01N15/02—Investigating particle size or size distribution
    • G01N2015/0294—Particle shape
    • G—PHYSICS
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    • 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
    • G01N2015/0687—Investigating concentration of particle suspensions in solutions, e.g. non volatile residue
    • 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
    • G01N15/10—Investigating individual particles
    • G01N2015/1006—Investigating individual particles for cytology
    • 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
    • G01N15/10—Investigating individual particles
    • G01N2015/1024—Counting particles by non-optical means
    • 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
    • G01N15/10—Investigating individual particles
    • G01N2015/1029—Particle size
    • 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
    • G01N15/10—Investigating individual particles
    • G01N2015/103—Particle shape
    • 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
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N2015/1486—Counting the particles
    • 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
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N2015/1493—Particle size
    • 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
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N2015/1497—Particle shape

Definitions

  • the invention relates to the technical field of particle detection, in particular to a method for detecting the concentration of micro-nano particles.
  • Particle concentration detection is a very important basic work. Particle concentration detection is widely used in cement, ceramics, pharmaceuticals, emulsions, paints, dyes, pigments, fillers, chemical products, catalysts, drilling mud, abrasives, lubricants, coal powder, mud sand, dust, cells, bacteria, food, additives, Pesticides, explosives, graphite, photosensitive materials, fuels, ink, metal and non-metal powders, calcium carbonate, kaolin, coal water slurry and other powdered materials.
  • impedance detection technology can realize the effective identification of individual particle characteristics and other obvious advantages, but the Coulter principle of impedance method can only measure the equivalent particle size of the particle, and cannot measure the potential and shape of the particle. At the same time, the measurement accuracy of the impedance method for small-sized particles is not high.
  • the colorimetric method uses the Lambert-Beer law to test the principle that the light absorption of suspended particles in a solution is proportional to the concentration, but the accuracy of this method is limited by the light absorption intensity, only when the absorbance is 1 to 2 The range is relatively accurate, and it is impossible to measure particles with lower concentrations.
  • Fluorescence and chemiluminescence methods both use specific luminescent groups to label target particles, and use luminescence intensity to detect the concentration of target particles.
  • the cost of detection equipment is relatively high, and on the other hand, there is the chemistry of luminescent groups.
  • the stability of the reagents is relatively high.
  • the invention provides a new single particle-based micro-nano particle concentration detection method, which has the advantages of high detection accuracy, low detection limit, and stable chemical reagents.
  • the present invention provides a method for detecting the concentration of micro-nano particles, the method comprising:
  • the above-mentioned label is polystyrene microspheres, magnetic beads, silicon spheres or micelles with specific functional groups or specific antigens or antibodies.
  • the above-mentioned specific functional groups are selected from biological enzymes and substrates, antigens and antibodies, ligands and receptors, and biotin and avidin.
  • the method for combining the above-mentioned micro/nano particles to be tested with the above-mentioned label includes specific antigen-antibody binding, special immunochemical reactions, specific chemical synthesis reactions, and specific binding of aptamers.
  • the manner in which the micro/nano particles to be measured are combined with the label includes one micro/nano particle to be measured combined with a label, one micro/nano particle to be measured combined with a plurality of labels, and a plurality of micro/nano particles to be measured are combined with a plurality of labels.
  • the micro-nano particles are combined with a label.
  • the above-mentioned micro/nano particles to be tested are selected from proteins, exosomes, and viruses.
  • the method for detecting the particle size, charge state, or particle morphology of the new particles and the micro/nano particles to be measured or the markers includes nanoparticle tracking (NTA), tunable resistance pulse sensing (TRPS). ) And single particle pulse technology and microscope imaging method.
  • NTA nanoparticle tracking
  • TRPS tunable resistance pulse sensing
  • the present invention provides a method for purification and concentration detection of micro-nano particles, the method comprising:
  • the sample containing the micro-nano particles to be tested is specifically combined with one or more magnetic bead markers in the solution, wherein the magnetic beads carry antigens or antibodies and aptamers that specifically bind to the micro-nano particles to be tested.
  • the magnetic beads carry antigens or antibodies and aptamers that specifically bind to the micro-nano particles to be tested.
  • Body or specific functional group so that at least one of the particle size, charge state, and particle morphology of the conjugate changes compared with the above-mentioned micro/nano particle to be tested or the above-mentioned magnetic bead marker;
  • the above-mentioned clear liquid is a liquid that does not contain particles that interfere with the detection of magnetic beads.
  • the above-mentioned micro/nano particles to be tested are selected from proteins, exosomes, and viruses.
  • the method for detecting the concentration of micro-nano particles of the present invention is based on single particle detection, combining the micro-nano particles to be measured with the marker to generate new particles.
  • Changes in state or particle morphology and separate counts to obtain the concentration of micro-nano particles to be tested that are bound to the label have the advantages of high detection accuracy, low detection limit, and stable chemical reagents.
  • Figure 1 is a histogram of particle size distribution in Example 1 of the present invention.
  • Example 2 is a scatter diagram of particle size-Zeta potential distribution in Example 2 of the present invention.
  • Fig. 3 is a scatter diagram of magnetic bead size-length-diameter ratio in embodiment 3 of the present invention.
  • Example 4 is a scatter plot of particle size-Zeta potential distribution in Example 4 of the present invention.
  • the present invention proposes a new method for detecting the concentration of micro-nano particles.
  • the micro-nano particles (target particles) to be detected are tested using markers. Specific binding is counted by the significant changes in particle size, charge state, or particle morphology to obtain the target particle concentration.
  • the present invention aims at various micro-nano particles that cannot be directly counted such as proteins, exosomes, viruses, etc., and can be tested and analyzed by the method of the present invention.
  • the detected micro-nano particles are exosomes (for example, kidney cell exosomes or lung tumor cell exosomes), Tau protein, troponin I, and the like.
  • These micro-nano particles can be derived from various tissues or body fluids of various organisms (for example, human body, etc.), for example, from kidney cell exosomes in urine, Tau protein from cerebrospinal fluid, and blood. The troponin I or lung tumor cell exosomes in the blood.
  • the particle size range that can be detected by the present invention is relatively wide, and particles ranging from nanometer level to micrometer level can be detected by the method of the present invention. It is preferable to detect particles of 5 nm to 5 ⁇ m.
  • a method for detecting the concentration of micro-nano particles includes:
  • a marker refers to a substance that can bind to the micro-nano particles to be tested and cause one or more characteristics of the micro-nano particles to be tested to change in particle size, charge state, and particle morphology.
  • the substance can be polystyrene microspheres, magnetic beads, silica balls or micelles with specific functional groups or specific antigens or antibodies, where the specific functional groups can be selected from biological enzymes and substrates, antigens and antibodies, ligands and receptors And biotin and avidin and other compounds or groups.
  • the markers are also micro-nano-level particles.
  • the label is polystyrene microspheres with specific binding antibodies, the particle size of which is hundreds of nanometers, such as 100nm, 150nm, 200nm, 300nm, 400nm, 500nm, etc., preferably 300nm ; Its concentration is based on the number in the test solution, which can be 1 ⁇ 10 10 /ml to 1 ⁇ 10 7 /ml, preferably 1 ⁇ 10 9 /ml.
  • the label is silicon microspheres with specific binding antibodies, the particle size of which is hundreds of nanometers, such as 100nm, 150nm, 200nm, 250nm, 300nm, 350nm, 400nm, 450nm, 500nm, etc. , Preferably 200nm; its concentration can be 1 ⁇ 10 10 /ml to 1 ⁇ 10 7 /ml, preferably 1 ⁇ 10 8 /ml according to the number in the detection solution.
  • the label is a magnetic bead with a specific binding antibody, and its particle size is tens of nanometers to hundreds of nanometers, such as 30nm, 50nm, 80nm, 100nm, 150nm, 200nm, 300nm, 400nm. , 500nm, etc., preferably 100nm; its concentration can be 1 ⁇ 10 10 /ml to 1 ⁇ 10 7 /ml, preferably 1 ⁇ 10 8 /ml according to the number in the detection solution.
  • the method for combining the micronanoparticle to be tested with the label includes specific antigen-antibody binding, special immunochemical reaction, specific chemical synthesis reaction, and specific binding of aptamer.
  • the method for combining the micro/nano particle to be measured with the label includes: combining one micro/nano particle to be measured with a label, combining one micro/nano particle to be measured with multiple labels, and multiple micro/nano particles to be measured.
  • the nanoparticle is bound to a label.
  • the new particles generated by the combination of the marker and the micro-nano particles to be tested have a change in one or more characteristics of the particle size, charge state, and particle morphology relative to the marker or the micro-nano particles to be tested.
  • the indicator to measure the particle size is generally the particle size, that is, the diameter of the particle, especially the average particle size;
  • the indicator to measure the charge state is generally the amount of charge or the electric mobility (ie the migration rate in electrophoresis);
  • the particle morphology for example, It can be spherical, ellipsoidal or other irregular shapes.
  • NTA nanoparticle tracing
  • TRPS Adjustable resistance pulse sensing
  • single particle pulse technology and microscope imaging method Typical but non-limiting examples include nanoparticle tracing (NTA) , Adjustable resistance pulse sensing (TRPS) and single particle pulse technology and microscope imaging method.
  • NTA nanoparticle tracing
  • TRPS Adjustable resistance pulse sensing
  • single particle pulse technology and microscope imaging method Typical but non-limiting examples include nanoparticle tracing (NTA) technology is used to detect the particle size and quantity of polystyrene microspheres and new particles after exosomes and polystyrene microspheres are combined.
  • tunable resistance pulse sensing is used to detect the particle size and quantity of silicon microspheres and new particles after Tau protein and silicon microspheres are combined.
  • the single particle pulse technology is used to detect the aspect ratio and the quantity of the nano magnetic beads and the new particles after the combination of troponin I and the nano magnetic beads.
  • the single particle pulse technique is used to detect the particle size and charge before and after the lung tumor cell exosomes in the blood sample are combined with the specific antibody, and obtain a particle size-Zeta potential distribution scatter diagram.
  • the present invention is based on the counting results of new particles and micro/nano particles to be measured or markers, and can calculate the concentration of micro/nano particles to be measured that are bound to the markers.
  • the calculation results are related to the counting results and reaction modes of various particles.
  • polystyrene microspheres (as markers) and exosomes (as micro-nano particles to be measured) are one-to-one
  • the binding method is combined, where the concentration of polystyrene microspheres is n, the number of polystyrene microspheres that are not bound to exosomes is N1, and the number of polystyrene microspheres that are bound to exosomes is N2.
  • the number of exosomes in the solution to be tested is: n*N2/(N1+N2).
  • the silicon microspheres (as the marker) and the Tau protein (as the micronanoparticles to be tested) are combined in a two-to-one manner, wherein the concentration of the silicon microspheres is n, and there is no combination with the Tau protein.
  • the number of bound silicon microspheres is N1
  • the number of silicon microspheres bound to Tau protein is N2
  • the number of Tau protein in the solution to be tested is: 2*n*N2/(N1+2*N2).
  • Another embodiment of the present invention provides a method for purification and concentration detection of micro-nano particles, including:
  • the sample containing the micro-nano particles to be tested is specifically combined with one or more magnetic bead markers in the solution, wherein the magnetic beads carry antigens or antibodies and aptamers that specifically bind to the micro-nano particles to be tested.
  • the magnetic beads carry antigens or antibodies and aptamers that specifically bind to the micro-nano particles to be tested.
  • Body or specific functional group so that at least one of the particle size, charge state, and particle morphology of the conjugate changes compared with the above-mentioned micro/nano particle to be tested or the above-mentioned magnetic bead marker;
  • the clear solution used to clean the magnetic beads should try to avoid interference with the detection of magnetic beads particles, for example, avoid containing magnetic particles.
  • the clear solution for washing the magnetic beads is a liquid that does not contain particles that interfere with the detection of the magnetic beads, for example, a physiological saline clear solution.
  • the method for detecting the concentration of micro-nano particles of the present invention is based on single particle detection, combining the micro-nano particles to be measured with the marker to generate new particles.
  • Changes in state or particle morphology and separate counts to obtain the concentration of micro-nano particles to be tested that are bound to the label have the advantages of high detection accuracy, low detection limit, and stable chemical reagents.
  • Centrifuge the urine sample of the nephritis patient with an ultracentrifuge take the supernatant liquid, and mix the supernatant liquid with the reagent containing antibody-modified polystyrene microspheres (particle size 300nm, concentration n 1 ⁇ 10 9 /ml)
  • the solution is mixed 1:1, and the polystyrene microspheres have antibodies that can specifically bind to the surface membrane proteins of kidney cell exosomes.
  • the mixture was reacted at 37°C for 20 minutes, and then the mixture was tested using NTA (nanoparticle tracing) technology to detect the particle size of polystyrene microspheres.
  • NTA nanoparticle tracing
  • the number of particles N1 in the range of 300 ⁇ 20nm is 11254, the number of particles in this range is the number of polystyrene microspheres that are not bound to exosomes; the number of particles N2 in the range of 340 ⁇ 460nm is 2430 The number of particles in this range is the number of polystyrene microspheres after being combined with exosomes.
  • the cerebrospinal fluid samples of patients with Alhuheimer's disease were filtered with porous resin, the filtered clear liquid was taken, and the clear liquid was combined with two kinds of antibody-modified silicon microspheres (both particle diameters of 200nm, concentration n of 1 ⁇ 10 8 /ml, Zeta potential-25mV) reagent solution is mixed 1:1, one of the silicon microspheres has an antibody that can specifically bind to Tau protein, and the other silicon microsphere has an antibody that can interact with Tau protein. An antibody that specifically binds to the other binding site.
  • the mixture was reacted at 37°C for 20 minutes, and then the mixture was tested using TRPS (tunable resistance pulse sensing) technology to detect the particle size and charge of the polystyrene microspheres.
  • TRPS tunable resistance pulse sensing
  • the number of particles N1 within the range of 200 ⁇ 5nm and the Zeta potential near -25mV is 5434.
  • the number of particles in this range is the number of silicon microspheres that are not bound to the Tau protein; those within the range of 380 ⁇ 10nm
  • the number of particles N2 is 630, and the number of particles in this range is the number of silicon microspheres after the double antibody is combined with the Tau protein.
  • the blood sample of the heart disease patient was mixed 1:1 with a reagent solution containing two antibody-modified magnetic beads.
  • One of the magnetic beads had an antibody capable of specifically binding to troponin I (cTnI).
  • the diameter is 100nm and the concentration n is 1 ⁇ 10 8 /ml.
  • Another magnetic bead has an antibody that can specifically bind to another site of troponin I (cTnI).
  • the particle size is 200nm and the concentration is n It is 1 ⁇ 10 8 /ml.
  • the mixture was reacted at 37°C for 20 minutes.
  • the number of particles N1 within the range of 200 ⁇ 5nm and the aspect ratio is around 1.0 is 4246, and the number of particles N2 within the range of 100 ⁇ 5nm and the aspect ratio is around 1.0 is 4240.
  • This range The number of particles is the number of magnetic beads that are not bound to troponin I; in the range of 290 ⁇ 10nm, the number of particles with an aspect ratio near 1.5 is 543, and the number of particles in this range is doubled with troponin I.
  • the total number of exosomes N1 was 4230, and the total exosomes concentration n was 5.34 ⁇ 10 8 /ml.
  • the number N2 of exosomes with a Zeta potential less than 20mV was 1014.
  • n*(M2/M1-N2/N1) 1.54 ⁇ 10 8 /ml.

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Abstract

一种微纳颗粒浓度的检测方法，包括：将待测微纳颗粒与一种或多种标记物相结合生成新颗粒，新颗粒在颗粒大小、电荷状态、颗粒形态中的至少一种与待测微纳颗粒或标记物相比发生变化；检测新颗粒与待测微纳颗粒或标记物的颗粒大小、电荷状态或颗粒形态，并对新颗粒与待测微纳颗粒或标记物分别进行计数获得各自计数结果，并基于计数结果计算得到与标记物发生结合的待测微纳颗粒的浓度。该方法具有检测准确度高、检测限低、化学试剂稳定的优点。

Description

一种微纳颗粒浓度的检测方法 技术领域
本发明涉及颗粒检测技术领域，尤其涉及一种微纳颗粒浓度的检测方法。
背景技术
液体中颗粒的浓度作为一项重要的物理参数，在医药、半导体、涂料墨水、过滤等行业标准里对颗粒浓度范围有明确的规定。颗粒浓度检测是一项非常重要的基础工作。颗粒浓度检测广泛应用于水泥、陶瓷、药品、乳液、涂料、染料、颜料、填料、化工产品、催化剂、钻井泥浆、磨料、润滑剂、煤粉、泥砂、粉尘、细胞、细菌、食品、添加剂、农药、炸药、石墨、感光材料、燃料、墨汁、金属与非金属粉末、碳酸钙、高岭土、水煤浆及其他粉状物料。
对于纳米颗粒浓度的测量，阻抗检测技术能实现单个颗粒特征的有效识别等明显优势，但是阻抗法库尔特原理只能测量微粒的等效粒径，无法测量粒子的电位和形态。同时阻抗法对小尺寸的微粒的测量精度不高。
比色法，是通过朗伯-比尔定律对溶液中悬浮颗粒对光的吸收度与浓度成正比的原理进行测试，但是这种方法的准确度受限于吸光强度，仅在吸光度为1～2的范围比较准确，无法测量较低浓度的微粒。
荧光法和化学发光法，都是通过特异性的发光基团对目标颗粒进行标记，通过发光强度来检测目标颗粒的浓度，一方面检测设备的成本比较高，另一方面存在发光基团的化学试剂的稳定性问题。
发明内容
本发明提供一种全新的基于单颗粒的微纳颗粒浓度的检测方法，具有检测准确度高、检测限低、化学试剂稳定的优点。
根据本发明的第一方面，本发明提供一种微纳颗粒浓度的检测方法，该方法包括：
将待测微纳颗粒与一种或多种标记物相结合生成新颗粒，上述新颗粒在颗粒大小、电荷状态、颗粒形态中的至少一种与上述待测微纳颗粒或上述标记物相比发生变化；
检测上述新颗粒与上述待测微纳颗粒或上述标记物的颗粒大小、电荷状态或颗粒形态，并对上述新颗粒与上述待测微纳颗粒或上述标记物分别进行计数获得各自计数结果，并基于上述计数结果计算得到与上述标记物发生结合的上述待测微纳颗粒的浓度。
在优选实施例中，上述标记物是带特异官能团或特异性抗原或抗体的聚苯乙烯微球、磁珠、硅球或胶束。
在优选实施例中，上述特异官能团选自生物酶与底物、抗原与抗体、配基与受体以及生物素与亲和素。
在优选实施例中，上述待测微纳颗粒与上述标记物相结合的方法包括特异性抗原抗体结合、特殊的免疫化学反应、特异性化学合成反应、适配体的特异性结合。
在优选实施例中，上述待测微纳颗粒与上述标记物相结合的方式包括一个待测微纳颗粒与一个标记物结合、一个待测微纳颗粒与多个标记物结合、多个待测微纳颗粒与一个标记物结合。
在优选实施例中，上述待测微纳颗粒选自蛋白质、外泌体、病毒。
在优选实施例中，上述检测上述新颗粒与上述待测微纳颗粒或上述标记物的颗粒大小、电荷状态或颗粒形态的方法包括纳米粒子示踪(NTA)、可调电阻脉冲感测(TRPS)和单粒子脉冲技术以及显微镜成像法。
根据本发明的第二方面，本发明提供一种微纳颗粒提纯并检测浓度的方法，该方法包括：
将包含待测微纳颗粒的样本与一种或多种磁珠标记物在溶液中进行特异性结合，其中磁珠上带有与上述待测微纳颗粒特异性结合的抗原或抗体、适配体或特异官能团，使得结合物在颗粒大小、电荷状态、颗粒形态中的至少一种与上述待测微纳颗粒或上述磁珠标记物相比发生变化；
将上一步产生的混合液置于磁场区域中，使上述磁珠被磁力吸附在磁场中的局部区域，再加入清液替换掉上述磁场区域中的混合液，使上述样本中的非待测微纳颗粒随着上述混合液的替换被清理掉，使得上述磁场区域中的清液中只含有磁珠颗粒；
检测上一步得到的清液中与待测微纳颗粒结合的磁珠和未能与待测微纳颗粒结合的磁珠在颗粒大小、电荷状态或颗粒形态上的变化，并对上述与待测微纳颗粒结合的磁珠和未能与待测微纳颗粒结合的磁珠分别进行计数获得各自计数结果，并基于上述计数结果计算得到与上述磁珠发生结合的上述待测微纳颗粒的浓度。
在优选实施例中，上述清液是不包含对磁珠的检测构成干扰的颗粒的液体。
在优选实施例中，上述待测微纳颗粒选自蛋白质、外泌体、病毒。
本发明的微纳颗粒浓度的检测方法，基于单颗粒进行检测，将待测微纳颗粒与标记物相结合生成新颗粒，通过检测新颗粒与待测微纳颗粒或标记物在颗粒大小、电荷状态或颗粒形态方面的变化和分别计数，得到与标记物发生结合的待测微纳颗粒的浓度，具有检测准确度高、检测限低、化学试剂稳定的优点。
附图说明
图1为本发明实施例1中的粒径分布柱状图；
图2为本发明实施例2中的粒径-Zeta电位分布散点图；
图3为本发明实施例3中的磁珠粒径-长径比散点图；
图4为本发明实施例4中的粒径-Zeta电位分布散点图。
具体实施方式
下面通过具体实施方式结合附图对本发明作进一步详细说明。在以下的实施方式中，很多细节描述是为了使得本发明能被更好的理解。然而，本领域技术人员可以毫不费力的认识到，其中部分特征在不同情况下是可以省略的，或者可以由其他材料、方法所替代。
另外，说明书中所描述的特点、操作或者特征可以以任意适当的方式结合形成各种实施方式。同时，方法描述中的各步骤或者动作也可以按照本领域技术人员所能显而易见的方式进行顺序调换或调整。因此，说明书和附图中的各种顺序只是为了清楚描述某一个实施例，并不意味着是必须的顺序，除非另有说明其中某个顺序是必须遵循的。
本发明针对现有技术检测微纳颗粒成本高、检测限高、准确度低的缺陷，提出了一种新的微纳颗粒浓度检测方法，对待检测的微纳颗粒(目标颗粒)使用标记物进行特异性结合，通过颗粒大小、电荷状态或颗粒形态上发生显著变化进行计数，得到目标颗粒浓度。
本发明针对蛋白质、外泌体、病毒等无法直接计数的各种微纳颗粒，可以通过本发明的方法进行测试分析。例如，在本发明实施例中，检测的微纳颗粒是外泌体(例如，肾脏细胞外泌体或肺肿瘤细胞外泌体)、Tau蛋白、肌钙蛋白I等。这些微纳颗粒可以来源于各种生物体(例如，人体等)的各种组织或体液，例如，来自于尿液中的肾脏细胞外泌体、来自于脑脊液中的Tau蛋白、来自于血液中的肌钙蛋白I或来自于血液中的肺肿瘤细胞外泌体等。
本发明能够检测的颗粒粒径范围比较宽泛，从纳米级别到微米级别的颗粒均可通过本发明的方法检测。优选检测5nm～5μm的颗粒。
根据本发明的实施例，一种微纳颗粒浓度的检测方法，包括：
将待测微纳颗粒与一种或多种标记物相结合生成新颗粒，上述新颗粒在颗粒大小、电荷状态、颗粒形态中的至少一种与上述待测微纳颗粒或上述标记物相比发生变化；
检测上述新颗粒与上述待测微纳颗粒或上述标记物的颗粒大小、电荷状态或颗粒形态，并对上述新颗粒与上述待测微纳颗粒或上述标记物分别进行计数获得各自计数结果，并基于上述计数结果计算得到与上述标记物发生结合的上述待测微纳颗粒的浓度。
本发明实施例中，标记物是指能够与待测微纳颗粒结合，并引起待测微纳颗粒在颗粒大小、电荷状态和颗粒形态中一种或多种特征发生变化的物质，这种标记物可以是带特异官能团或特异性抗原或抗体的聚苯乙烯微球、磁珠、硅球或胶束等，其中特异官能团可以选自生物酶与底物、抗原与抗体、配基与受体以及生物素与亲和素等化合物或基团。
本发明实施例中，一般而言，标记物也是微纳米级别的颗粒。例如，在本发明的一个实施例中，标记物是带特异性结合抗体的聚苯乙烯微球，其粒径是数百纳米，例如100nm、150nm、200nm、300nm、400nm、500nm等，优选300nm；其浓度按照在检测液中个数计，可以是1×10 10/ml至1×10 7/ml，优选1×10 9/ml。在本发明的另一个实施例中，标记物是带特异性结合抗体的硅微球，其粒径是数百纳米，例如100nm、150nm、200nm、 250nm、300nm、350nm、400nm、450nm、500nm等，优选200nm；其浓度按照在检测液中个数计，可以是1×10 10/ml至1×10 7/ml，优选1×10 8/ml。在本发明的又一个实施例中，标记物是带特异性结合抗体的磁珠，其粒径是几十纳米至几百纳米，例如30nm、50nm、80nm、100nm、150nm、200nm、300nm、400nm、500nm等，优选100nm；其浓度按照在检测液中个数计，可以是1×10 10/ml至1×10 7/ml，优选1×10 8/ml。
本发明实施例中，待测微纳颗粒与标记物相结合的方法包括特异性抗原抗体结合、特殊的免疫化学反应、特异性化学合成反应、适配体的特异性结合。
本发明实施例中，待测微纳颗粒与标记物相结合的方式包括：一个待测微纳颗粒与一个标记物结合、一个待测微纳颗粒与多个标记物结合、多个待测微纳颗粒与一个标记物结合。
本发明实施例中，标记物与待测微纳颗粒结合生成的新颗粒相对于标记物或待测微纳颗粒而言，在颗粒大小、电荷状态和颗粒形态中一种或多种特征发生变化。其中，衡量颗粒大小的指标一般是粒径，即颗粒的直径，尤其是平均粒径；衡量电荷状态的指标一般是电荷量或电迁移率(即电泳中的迁移速率)；颗粒形态，例如，可以是球形、椭球型或其它不规则形状等。
本发明实施例中，检测生成的新颗粒与待测微纳颗粒或标记物的颗粒大小、电荷状态或颗粒形态的方法有多种，典型但非限定性的例子包括纳米粒子示踪(NTA)、可调电阻脉冲感测(TRPS)和单粒子脉冲技术以及 显微镜成像法。例如，在本发明一个实施例中，使用纳米粒子示踪(NTA)技术检测聚苯乙烯微球以及外泌体与聚苯乙烯微球结合后的新颗粒的粒径及其数量。在本发明另一个实施例中，使用可调电阻脉冲感测(TRPS)检测硅微球以及Tau蛋白与硅微球结合后的新颗粒的粒径及其数量。在本发明又一个实施例中，使用单粒子脉冲技术检测纳米磁珠以及肌钙蛋白I与纳米磁珠结合后的新颗粒的长径比及其数量。在本发明再一个实施例中，使用单粒子脉冲技术检测血液样本中肺肿瘤细胞外泌体与特异性抗体结合前后的粒径和电荷并得到粒径-Zeta电位分布散点图。
本发明基于新颗粒和待测微纳颗粒或标记物的计数结果，能够计算得到与标记物发生结合的待测微纳颗粒的浓度。计算结果与各种颗粒的计数结果和反应模式相关，例如，在本发明一个实施例中，聚苯乙烯微球(作为标记物)与外泌体(作为待测微纳颗粒)按照一对一结合的方式结合，其中聚苯乙烯微球的浓度是n，没有与外泌体结合的聚苯乙烯微球的数量是N1，与外泌体结合之后的聚苯乙烯微球的数量是N2，那么待检测溶液中外泌体的数量为：n*N2/(N1+N2)。在本发明另一个实施例中，硅微球(作为标记物)与Tau蛋白(作为待测微纳颗粒)按照二对一结合的方式结合，其中硅微球的浓度是n，没有与Tau蛋白结合的硅微球的数量是N1，与Tau蛋白结合的硅微球的数量是N2，那么待检测溶液中Tau蛋白的数量为：2*n*N2/(N1+2*N2)。
本发明的另一个实施例提供一种微纳颗粒提纯并检测浓度的方法，包括：
将包含待测微纳颗粒的样本与一种或多种磁珠标记物在溶液中进行特异性结合，其中磁珠上带有与上述待测微纳颗粒特异性结合的抗原或抗体、适配体或特异官能团，使得结合物在颗粒大小、电荷状态、颗粒形态中的至少一种与上述待测微纳颗粒或上述磁珠标记物相比发生变化；
将上一步产生的混合液置于磁场区域中，使上述磁珠被磁力吸附在磁场中的局部区域，再加入清液替换掉上述磁场区域中的混合液，使上述样本中的非待测微纳颗粒随着上述混合液的替换被清理掉，使得上述磁场区域中的清液中只含有磁珠颗粒；
检测上一步得到的清液中与待测微纳颗粒结合的磁珠和未能与待测微纳颗粒结合的磁珠在颗粒大小、电荷状态或颗粒形态上的变化，并对上述与待测微纳颗粒结合的磁珠和未能与待测微纳颗粒结合的磁珠分别进行计数获得各自计数结果，并基于上述计数结果计算得到与上述磁珠发生结合的上述待测微纳颗粒的浓度。
本发明实施例中，用于清洗磁珠的清液中要尽量避免对磁珠颗粒的检测构成干扰，例如避免含有磁性颗粒物。在本发明的一个实施例中，清洗磁珠的清液是不包含对磁珠的检测构成干扰的颗粒的液体，例如，生理盐水清液。
本发明的微纳颗粒浓度的检测方法，基于单颗粒进行检测，将待测微纳颗粒与标记物相结合生成新颗粒，通过检测新颗粒与待测微纳颗粒或标记物在颗粒大小、电荷状态或颗粒形态方面的变化和分别计数，得到与标记物发生结合的待测微纳颗粒的浓度，具有检测准确度高、检测限低、化 学试剂稳定的优点。
以下通过具体实施例详细说明本发明的技术方案，应当理解，实施例仅是示例性的，不能理解为对本发明保护范围的限制。
实施例1
用带特异性结合抗体的聚苯乙烯微球检测尿液中肾脏细胞外泌体浓度
将肾炎病人的尿液样本用超速离心机离心，取上部清液，将清液与含有经过抗体修饰的聚苯乙烯微球(粒径为300nm、浓度n为1×10 9/ml)的试剂溶液进行1：1混合，聚苯乙烯微球上带有能够与肾脏细胞外泌体的表面膜蛋白进行特异性结合的抗体。混合液在37℃温度下反应20分钟，之后对混合液使用NTA(纳米粒子示踪)技术进行测试，实现对聚苯乙烯微球的粒径检测。得到如图1所示的粒径分布柱状图。
测试到在300±20nm范围内的粒子数N1为11254个，这个范围的粒子数为没有与外泌体结合的聚苯乙烯微球的数量；在340～460nm范围内的粒子数N2为2430个，这个范围内的粒子数为与外泌体结合之后的聚苯乙烯微球的数量。
原样本中的肾细胞外泌体数量为：n*N2/(N1+N2)＝1.77×10 8/ml。
实施例2
用带特异性结合抗体的硅微球检测脑脊液中Tau蛋白的浓度
将阿尔胡海默症病人的脑脊液样本用多孔树脂进行过滤，取过滤后的 清液，将清液与含有两种经过抗体修饰的硅微球(粒径均为200nm、浓度n均为1×10 8/ml、Zeta电位-25mV)的试剂溶液进行1：1混合，其中一种硅微球上带有能够与Tau蛋白进行特异性结合的抗体，另一种硅微球上带有能够与Tau蛋白的另外一个结合位点进行特异性结合的抗体。混合液在37℃温度下反应20分钟，之后对混合液使用TRPS(可调电阻脉冲感测)技术进行测试，实现对聚苯乙烯微球的粒径和电荷的检测。得到如图2所示的粒径-Zeta电位分布散点图。
测试到在粒径200±5nm范围内、Zeta电位在-25mV附近的粒子数N1为5434个，这个范围的粒子数为没有与Tau蛋白结合的硅微球的数量；在380±10nm范围内的粒子数N2为630个，这个范围内的粒子数为与Tau蛋白进行双抗结合之后的硅微球的数量。
原样本中的Tau蛋白数量为：2*n*N2/(N1+2*N2)＝1.88×10 7/ml。
实施例3
使用带特异性结合抗体的磁珠检测血液中肌钙蛋白I的浓度
将心脏病人的血液样本与含有两种经过抗体修饰的磁珠的试剂溶液进行1：1混合，其中一种磁珠上带有能够与肌钙蛋白I(cTnI)进行特异性结合的抗体，粒径为100nm、浓度n为1×10 8/ml，另一种磁珠上带有能够与肌钙蛋白I(cTnI)的另外一个位点进行特异性结合的抗体，粒径为200nm、浓度n为1×10 8/ml。混合液在37℃温度下反应20分钟。
将混合液倒入试管中，将试管置于强磁场中，使磁珠富集在试管的下部；用移液器移走试管上部区域的液体，撤去磁场，加入生理盐水清液进 行混匀。再将试管置于强磁场中，重复上述步骤，直至试管中的生理盐水只有高纯度的磁珠。
将上述含有磁珠的生理盐水使用单粒子脉冲技术进行测试，得到如图3所示的磁珠粒径-长径比散点图。
测试到在粒径200±5nm范围内、长径比在1.0附近的粒子数N1为4246个，在粒径100±5nm范围内、长径比在1.0附近的粒子数N2为4240个，这个范围的粒子数为没有与肌钙蛋白I结合的磁珠的数量；在290±10nm范围，长径比在1.5附近的粒子数N3为543个，这个范围的粒子数为与肌钙蛋白I进行双抗结合之后的磁珠的数量。
原样本中的肌钙蛋白I数量为：2*n*N3/(N1+N2+2*N3)＝1.13×10 7/ml。
实施例4
用特异性抗体测量血液样本中肺肿瘤细胞外泌体的浓度
将肺肿瘤病人的血液样本用超速离心机离心，取上部清液，清液中不含细胞、血小板等大颗粒。将一部分使用单粒子脉冲技术技术进行测试，实现对外泌体的粒径和电荷的检测。得到如图4中左图所示的粒径-Zeta电位分布散点图。
测得总的外泌体个数N1为4230个，测得总的外泌体浓度n为5.34×10 8/ml。Zeta电位小于20mV的外泌体的数量N2为1014个。
将清液中加入含肺细胞外泌体表面膜蛋白抗体的试剂溶液(试剂中的抗体过量，抗体带负的Zeta电位)。混合液在37℃温度下反应20分钟， 之后对混合液使用单粒子脉冲技术进行测试，实现对外泌体的粒径和电荷的检测。得到如图4中右图所示的粒径-Zeta电位分布散点图。肺细胞外泌体与抗体结合之后电荷变小，测得总的外泌体个数M1为3802个，Zeta电位小于20mV的外泌体的数量M2为2011个，Zeta电位小于20mV的外泌体增加的数量为肺细胞外泌体的数量。
原样本中的肺细胞外泌体数量为：n*(M2/M1-N2/N1)＝1.54×10 8/ml。
以上应用了具体个例对本发明进行阐述，只是用于帮助理解本发明，并不用以限制本发明。对于本发明所属技术领域的技术人员，依据本发明的思想，还可以做出若干简单推演、变形或替换。

Claims (10)

  1. 一种微纳颗粒浓度的检测方法，其特征在于，所述方法包括：
    将待测微纳颗粒与一种或多种标记物相结合生成新颗粒，所述新颗粒在颗粒大小、电荷状态、颗粒形态中的至少一种与所述待测微纳颗粒或所述标记物相比发生变化；
    检测所述新颗粒与所述待测微纳颗粒或所述标记物的颗粒大小、电荷状态或颗粒形态，并对所述新颗粒与所述待测微纳颗粒或所述标记物分别进行计数获得各自计数结果，并基于所述计数结果计算得到与所述标记物发生结合的所述待测微纳颗粒的浓度。
  2. 根据权利要求1所述的方法，其特征在于，所述标记物是带特异官能团或特异性抗原或抗体的聚苯乙烯微球、磁珠、硅球或胶束。
  3. 根据权利要求2所述的方法，其特征在于，所述特异官能团选自生物酶与底物、抗原与抗体、配基与受体以及生物素与亲和素。
  4. 根据权利要求1所述的方法，其特征在于，所述待测微纳颗粒与所述标记物相结合的方法包括特异性抗原抗体结合、特殊的免疫化学反应、特异性化学合成反应、适配体的特异性结合。
  5. 根据权利要求1所述的方法，其特征在于，所述待测微纳颗粒与所述标记物相结合的方式包括一个待测微纳颗粒与一个标记物结合、一个待测微纳颗粒与多个标记物结合、多个待测微纳颗粒与一个标记物结合。
  6. 根据权利要求1所述的方法，其特征在于，所述待测微纳颗粒选自 蛋白质、外泌体、病毒、聚苯乙烯微球、磁珠、硅球或胶束。
  7. 根据权利要求1所述的方法，其特征在于，所述检测所述新颗粒与所述待测微纳颗粒或所述标记物的颗粒大小、电荷状态或颗粒形态的方法包括纳米粒子示踪(NTA)、可调电阻脉冲感测(TRPS)和单粒子脉冲技术以及显微镜成像法。
  8. 一种微纳颗粒提纯并检测浓度的方法，其特征在于，所述方法包括：
    将包含待测微纳颗粒的样本与一种或多种磁珠标记物在溶液中进行特异性结合，其中磁珠上带有与所述待测微纳颗粒特异性结合的抗原或抗体、适配体或特异官能团，使得结合物在颗粒大小、电荷状态、颗粒形态中的至少一种与所述待测微纳颗粒或所述磁珠标记物相比发生变化；
    将上一步产生的混合液置于磁场区域中，使所述磁珠被磁力吸附在磁场中的局部区域，再加入清液替换掉所述磁场区域中的混合液，使所述样本中的非待测微纳颗粒随着所述混合液的替换被清理掉，使得所述磁场区域中的清液中只含有磁珠颗粒；
    检测上一步得到的清液中与待测微纳颗粒结合的磁珠和未能与待测微纳颗粒结合的磁珠在颗粒大小、电荷状态或颗粒形态上的变化，并对所述与待测微纳颗粒结合的磁珠和未能与待测微纳颗粒结合的磁珠分别进行计数获得各自计数结果，并基于所述计数结果计算得到与所述磁珠发生结合的所述待测微纳颗粒的浓度。
  9. 根据权利要求8所述的方法，其特征在于，所述清液是不包含对磁珠的检测构成干扰的颗粒的液体。
  10. 根据权利要求8所述的方法，其特征在于，所述待测微纳颗粒选自蛋白质、外泌体、病毒、聚苯乙烯微球、磁珠、硅球或胶束。
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