CN108593910B - Particle detection system and method based on microsphere carrier - Google Patents
Particle detection system and method based on microsphere carrier Download PDFInfo
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- 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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—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
Abstract
The invention relates to a particle detection system and a method based on a microsphere carrier, the system comprises a heating unit and a sample chamber unit, wherein immune microsphere fluid combined with target biomolecules is loaded in the sample chamber unit, the immune microspheres are prepared by fixing antibodies or aptamers on the surfaces of microspheres, the immune microspheres combined with the target biomolecules are marked by fluorescence, after the sample chamber unit is heated by the heating unit, thermophoresis effect is generated in the sample chamber unit, and the exosomes are gathered at the lower temperature side in the sample chamber unit so as to amplify the fluorescence signals marked on the immune microspheres combined with the target biomolecules in the sample chamber for detection. The biomacromolecules such as free protein, nucleic acid and the like are modified on the surface of a micron-sized sphere with an antibody or aptamer capable of being specifically combined with target protein and nucleic acid to obtain an immune microsphere, so that free particles are converged and detected after being converged.
Description
Technical Field
The invention relates to the field of micro-nano particle detection, in particular to a particle detection system and method based on a microsphere carrier.
Background
In the prior art, micro-nano particles are detected to measure the size, shape, concentration, activity and the like of particles, and the method is widely applied to the subjects of hematology, immunology, molecular biology and the like. In the prior art, a flow type particle detection method is commonly used for detecting micro-nano particles, which is a technology for quantitatively analyzing and sorting the particle particles in liquid one by one, and the coulter principle adopted in the detection is as follows: when the particles suspended in the electrolyte pass through the small holes along with the electrolyte, the particles replace the electrolyte with the same volume, the resistance between the inner electrode and the outer electrode of the small holes is instantaneously changed in a constant current designed circuit, potential pulses are generated, and the size and the frequency of pulse signals are in direct proportion to the size and the number of the particles. Sample focusing is a key technology of flow type particle detection, and in the current detection, sample liquid is focused through the action of external force. Focusing is further divided into focusing by a sheath fluid and focusing without a sheath fluid.
The sheath fluid is focused as in the microfluidic particle analyzer and the manufacturing method disclosed in chinese patent 201210482142.7, wherein the sample fluid is injected from the sample fluid inlet by the pressure of an external syringe pump, the sheath fluid is injected from the sheath fluid inlet, then the sample fluid and the two sheath fluids flow into the sheath fluid convergence region at the same time, and the particle particles in the sample fluid are sandwiched by the sheath fluid aggregation to be linearly arranged and flow into the detection region for detection. In the method, two sheath flows and sample liquid both need a driving source, and a mode of controlling three pipelines by one motor is adopted, so that the equipment becomes huge, the cost is also improved, more importantly, as the chip needs to be replaced when the detection is carried out each time, the three channels and the motor need to be connected again when the detection is carried out each time, the tightness problem of the connection part can influence the pressure of the three channels, the focusing effect is poor, and the test result is not accurate enough.
For example, a conical focusing structure is adopted in a microfluidic chip structure for a flow type particle analyzer and a manufacturing method thereof disclosed in chinese patent 201310283051.5, and the conical focusing structure is considered to have a focusing effect similar to that of a conventional sheath fluid flow system, so that particle particles flow into a microchannel singly, the microchannel binds the particles through the channel and enables the particles to pass through a detection area singly, and inaccuracy of a detection result is caused under a detection condition of a high-concentration sample.
In the two technical schemes for detecting the micro-nano particles, on one hand, potential pulses are generated through electrolysis, and the nano particles are separated and detected by adopting an electrochemical method to form a flow beam containing the micro-nano particles, so that the required sample amount is extremely large; on the other hand, the flow direction and the accumulation direction of the micro-nano particles are limited by a driving source such as a motor and a single channel with a fixed structure, and the external force acts on the fluid in the process of applying the external force and limiting the channel, so that the force applied to the micro-nano particles is often uncontrollable.
In particular, the detection of micro-nano biological particles such as exosome, which is a membrane vesicle secreted by cells and used for intercellular communication, has become an emerging biomarker for non-invasive tumor diagnosis in recent years because it contains proteins and genetic materials related to the mother cells.
The prior art generally adopts: first, enzyme-linked immunosorbent assay, ELISA, refers to a qualitative and quantitative detection method in which soluble antigen or antibody is bound to a solid-phase carrier such as polystyrene, and immunoreaction is carried out by utilizing the binding specificity of antigen and antibody, wherein, in the measurement, a sample to be detected (antibody or antigen in the sample to be detected) and enzyme-labeled antigen or antibody react with the antigen or antibody on the surface of the solid-phase carrier according to different steps; the antigen-antibody complex formed on the solid phase carrier is separated from other substances by washing, and finally the enzyme quantity bound on the solid phase carrier is in a certain proportion to the quantity of the detected substance in the specimen. After the substrate of the enzyme reaction is added, the substrate is catalyzed by the enzyme to be changed into a colored product, and the amount of the product is directly related to the amount of the detected substance in the sample, so that the qualitative or quantitative analysis can be carried out according to the shade of the color reaction.
Secondly, Western blotting, which is Western Blot, has the basic principle that a cell or biological tissue sample treated by gel electrophoresis is stained by a specific antibody; information on the expression of a specific protein in the analyzed cell or tissue is obtained by analyzing the location and depth of staining.
In the two technical schemes, on one hand, special equipment and methods are needed for separating and accumulating samples; on the other hand, the detection method needs to adopt a large amount of samples, and the process of detecting canceration by aiming at serum is not adaptive.
European patent publication No. EP2783747 also discloses a liquid mixing method using convection heat flow, but by passing a liquid through a capillary and placing the capillary in parallel and/or anti-parallel guidance by gravity, since the liquid is guided in the capillary, the liquid in the particles cannot move in the same direction as intended and cannot be accumulated, and thus, the detection of fine particles cannot be performed by this guiding method. In particular, free particles, such as free proteins, nucleic acids, and other biological macromolecules, cannot be accumulated and detected.
Disclosure of Invention
The invention aims to provide a particle detection system and a particle detection method based on a microsphere carrier, which are used for overcoming the technical defect that the accumulation detection of free particles cannot be carried out in the prior art.
In order to achieve the above object, the present invention provides a particle detection system based on a microsphere carrier, comprising a heating unit and a sample chamber unit, wherein,
the heating unit is used for heating the sample in the sample chamber unit;
the kit comprises a sample chamber unit, a heating unit, a sample chamber unit and an exosome, wherein an immune microsphere fluid combined with target biomolecules is loaded in the sample chamber unit, the immune microsphere fluid is prepared by fixing antibodies or aptamers on the surface of microspheres, the immune microsphere combined with the target biomolecules is marked by fluorescence, after the sample chamber unit is heated by the heating unit, thermophoresis effect is generated in the sample chamber unit, and the exosome is gathered at the lower side of the temperature in the sample chamber unit so as to amplify a fluorescence signal marked on the immune microsphere combined with the target biomolecules in the sample chamber for detection.
Further, the system also comprises a signal acquisition unit, wherein the signal acquisition unit acquires the amplified fluorescent signal to obtain the abundance of the target biomolecule combined on the surface of the immune microsphere, and the detection result is obtained by respectively combining various aptamers with different target proteins on the surface of the immune microsphere combined with the target biomolecule.
Further, the sample chamber unit comprises a closed sample chamber loaded with immune microspheres bound with target biomolecules and used for providing a space for generating thermophoretic effect, the sample chamber comprises: and the second heat conduction surface is used for sealing the sample chamber and accumulating the micro-nano particles.
Further, the temperature near the second heat conducting surface is lower than the temperature of the micro-nano particle fluid, so that a temperature difference is generated between the second heat conducting surface and the immune microsphere fluid combined with the target biological molecules, a thermophoresis effect is generated, and the immune microspheres combined with the target biological molecules are driven to move towards the second heat conducting surface in a directional mode.
Further, the heating unit is a laser device arranged on the outer side of the sample chamber unit, the laser device irradiates the sample chamber unit, and the light beam sequentially passes through the immune microsphere fluid combined with the target biological molecules and the second heat conduction surface so as to generate a thermophoresis effect on the immune microsphere solution combined with the target biological molecules.
Further, the sample chamber further comprises: the first heat conducting surface is used for sealing the sample chamber, and the second heat conducting surface and the first heat conducting surface can both allow light beams to pass through.
Furthermore, the second heat conduction surface is made of a transparent material and is made of a sapphire material;
the first heat conduction surface is any one or combination of glass, polymethyl methacrylate, polydimethylsiloxane and sapphire.
Further, the microspheres are polystyrene microspheres.
The invention also provides a particle detection method based on the microsphere carrier, which comprises the following steps:
preparing immune microspheres, and incubating the microspheres and the antibody or aptamer together to fix the antibody or aptamer on the surfaces of the microspheres to obtain the immune microspheres;
incubating the immune microspheres and a sample to be detected, and specifically binding target proteins or nucleic acids in the sample to be detected with antibodies or aptamers on the immune microspheres so as to fix the immune microspheres;
combining the prepared immune microsphere combined with the target biomolecule with an antibody or an aptamer carrying a fluorescent group, and marking the target biomolecule on the immune microsphere with fluorescence;
the immune microsphere sample combined with the target biological molecules loaded in the sample chamber unit is heated, a thermophoresis effect is generated in the sample chamber unit, so that the immune microspheres combined with the target biological molecules are gathered on the low-temperature side in the sample chamber unit, and signals are amplified due to fluorescent label enrichment for detection.
Further, the immune microspheres combined with the target biomolecules accumulated on the low-temperature side in the sample chamber unit are used for collecting corresponding index information of the immune microspheres combined with the target biomolecules and analyzing the corresponding indexes.
Compared with the prior art, the micro-nano detection system has the beneficial effects that the micro-nano detection system heats one direction of the sample chamber unit where the micro-nano particles are located, thermophoresis effect and convection are introduced, so that the sample chamber unit generates temperature difference, low temperature is generated at one side far away from the heating unit, and the thermophoresis effect enables the micro-nano particles in the sample to migrate and accumulate to the sample chamber unit so as to complete the accumulation of the micro-nano particles; meanwhile, the sample liquid is heated and expanded to generate buoyancy, so that convection is generated in the sample chamber unit, and the convection direction points to the heating area of the sample chamber unit from the periphery in the low-temperature area of the sample chamber unit, so that the accumulation of micro-nano particles is further promoted.
In particular, in the invention, biological macromolecules such as free protein, nucleic acid and the like or biological macromolecules such as protein, nucleic acid and the like which are not exposed on the surface in exosome are modified on the surface of a micron-sized sphere to obtain an immune microsphere, and the immune microsphere is incubated with a sample containing the target biological macromolecules, combined with the target biological macromolecules and labeled with fluorescence so as to converge free particles or the target biological macromolecules which are not exposed on the surface and detect the converged particles.
Further, the system incubates a sample to be detected containing the exosomes and a fluorescence-labeled aptamer or antibody, and the exosomes are labeled with fluorescence through the specific binding of the aptamer or antibody and the exosome surface protein; the incubated sample is placed into an upper and lower transparent sample chamber and is placed on a fluorescence microscope objective table for observation, an infrared laser irradiates and penetrates through the action of the sample chamber and the sample, the exosome in the sample is highly enriched at the laser light point at the bottom of the sample chamber through the thermophoresis action, so that the fluorescence height of the exosome is amplified, and the abundance of the surface protein of a certain exosome is detected through the fluorescence intensity.
Furthermore, the system adopts laser to irradiate the sample chamber for heating, and the transparent heat-conducting surfaces with different heat-conducting properties are arranged on the opposite side surfaces of the sample chamber, so that temperature difference is generated between the two heat-conducting surfaces to generate thermophoresis effect, and micro-nano particles are driven to directionally move from the second heat-conducting surface with the first heat-conducting surface and the lower temperature. Especially, the light beam heating is adopted, other auxiliary equipment is not needed, and only the transparent heat conducting surfaces are arranged on the upper portion and the lower portion of the sample chamber. Moreover, the force of the micro-nano particles under the thermophoresis effect is in direct proportion to the square of the particle diameter and is irrelevant to the quantity of the micro-nano particles, so that the aggregation and detection can be completed only by micro-nano particles, the sample dosage of 0.1 microliter is only needed for exosomes, the operation is convenient, special instruments are not needed, and the method is not needed for sample pretreatment and exosome purification and is generally used for aptamers and antibodies; the protein is not limited to exosomes, and other micro-nano biological particles such as extracellular vesicles and cells can be used.
Particularly, the system and the method can finish measurement at a specific temperature, are not limited by specific temperature, and only need to generate temperature difference to accumulate particles; measurements can also be done in a variety of different solution environments, including the complex detergent environments required for studying membrane proteins; it is also possible to use a variety of different sized molecules: for example, ions, nucleic acid fragments, nucleosomes and liposomes are detected, and when the detection is specifically carried out, the system can adjust parameters such as the temperature difference, the height between the upper and lower heat conducting surfaces, the type of fluid and the frequency of laser irradiation according to the physical properties of particles and the size of the particles, and the adjustment of all the parameters can realize quantitative adjustment, and is accurate in control and convenient to adjust.
The invention is based on the thermophoresis effect to accumulate particles, has no limit on a loading container of micro-nano particles, and is particularly easy to accumulate the particles under the thermophoresis effect in a container with larger volume without considering the guide of carrier containers such as a capillary tube and the like.
Drawings
FIG. 1 is a structural block diagram of a micro-nano particle detection system of the invention;
FIG. 2 is a block diagram of an exosome-based signal detection flow of the present invention;
FIG. 3 is a comparative spectrum of exosomes of example 1 of the present invention before and after the experiment;
FIG. 4 is a schematic diagram showing the surface protein maps and the corresponding surface protein expression levels of the exosomes of example 2 of the present invention after detection;
FIG. 5 is a diagram showing the expression levels of various types of proteins in serum exosomes of various cancer patients in healthy humans according to example 2 of the present invention;
FIG. 6 is a graph showing fluorescence measurement gray scale values of fluorescent polystyrene microspheres of different diameters according to example 3 of the present invention;
FIG. 7 is a diagram showing the expression levels of 11 protein markers in the serum of ovarian cancer patients and healthy persons according to example 4 of the present invention;
FIG. 8 is a graphical representation of the accuracy of the 11 different markers and their sum as criteria for distinguishing between cancer and health in example 4 of the present invention.
Detailed Description
The above and further features and advantages of the present invention are described in more detail below with reference to the accompanying drawings.
Preferred embodiments of the present invention are described below with reference to the accompanying drawings. It should be understood by those skilled in the art that these embodiments are only for explaining the technical principle of the present invention, and are not intended to limit the scope of the present invention.
It should be noted that in the description of the present invention, the terms of direction or positional relationship indicated by the terms "upper", "lower", "left", "right", "inner", "outer", etc. are based on the directions or positional relationships shown in the drawings, which are only for convenience of description, and do not indicate or imply that the device or element must have a specific orientation, be constructed in a specific orientation, and be operated, and thus, should not be construed as limiting the present invention. Furthermore, the terms "first" and "second" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance.
Furthermore, it should be noted that, in the description of the present invention, unless otherwise explicitly specified or limited, the terms "mounted," "connected," and "connected" are to be construed broadly, and may be, for example, fixedly connected, detachably connected, or integrally connected; can be mechanically or electrically connected; they may be connected directly or indirectly through intervening media, or they may be interconnected between two elements. The specific meanings of the above terms in the present invention can be understood by those skilled in the art according to specific situations.
Referring to fig. 1, which is a block diagram of a micro-nano particle detection system according to the present invention, the system of the present embodiment includes a heating unit 1, a sample chamber unit 2, and a signal collecting unit 4, wherein the heating unit 1 is disposed at an outer side of the sample chamber unit 2, and is configured to heat a sample in the sample chamber unit 2; micro-nano particles are loaded in the sample chamber unit 2, and after the heating unit 1 heats the sample chamber unit 2, a thermophoresis effect is generated in the sample chamber unit 2 so as to converge the micro-nano particles on one side, far away from the heating unit 1, in the sample chamber unit 2; and the signal acquisition unit 4 is used for acquiring related signal information of the micro-nano particles after the micro-nano particles in the sample bin unit 2 are accumulated, and correspondingly analyzing the corresponding micro-nano particles. The system utilizes the thermophoresis effect, namely the directional migration of an object under the action of temperature gradient, and generates a temperature gradient field at the local part of a sample through the irradiation of infrared laser, so that exosomes in the sample are migrated to a lower temperature place. By heating one direction of the sample chamber unit 2 where the micro-nano particles are located, thermophoresis effect and convection are introduced, so that a micro-nano particle fluid in temperature difference generated by the sample chamber unit 2 generates low-temperature difference with one side of the sample chamber unit 2, the temperature of one side of the sample chamber unit 2 is lower than that of the micro-nano particle fluid, and the micro-nano particles in a sample are migrated and accumulated to the low-temperature side of the sample chamber unit 2 through the thermophoresis effect; meanwhile, a convection current is generated in the sample chamber unit 2 due to buoyancy generated by the expansion of the sample liquid fluid by heat. In the low temperature region of the sample chamber unit 2, the convection direction points to the heating region of the sample chamber unit 2 from the periphery, and as indicated by the arrow in fig. 1, the convection direction acts as a conveyor belt to gather the surrounding micro-nano particles on the low temperature side of the sample chamber unit 2, thereby accumulating the micro-nano particles. Therefore, the particles are accumulated based on the thermophoresis effect, the loading container of the micro-nano particles is not limited, and particularly in a container with a larger volume, the particles are easier to accumulate under the thermophoresis effect without considering the guidance of a carrier container such as a capillary tube.
Specifically, the heating unit 1 of the present embodiment is a laser that is disposed outside the sample chamber unit 2 and irradiates the inside of the sample chamber unit 2 to generate a circular heating region therein, but the heating region may be linear or in other forms. It can be understood by those skilled in the art that the heating method is not limited to laser irradiation, the laser irradiation direction only needs to ensure the generation of a heat source, and the power selection depends on the irradiation direction, the spot diameter, the wavelength and other factors, and can be changed according to the actual micro-nano particles and the use environment.
Specifically, the sample chamber unit 2 includes a sealed sample chamber 24 for loading the micro-nano particle sample and providing a space for generating a thermophoresis effect, the sample chamber 24 includes a first heat conduction surface 21 for sealing the sample chamber 24, and a second heat conduction surface 22 for sealing the sample chamber 24, in this embodiment, a temperature difference is generated between the micro-nano particle fluid loaded in the sample chamber 24 and the second heat conduction surface 22 to generate a thermophoresis effect, and the micro-nano particles are driven to directionally move from the micro-nano particle fluid to the second heat conduction surface 22. Therefore, in this embodiment, the temperature near the second heat conduction surface 22 is lower than the temperature of the micro-nano particle fluid.
In this embodiment, adopt the laser instrument to heat sample room 24, first heat conduction face 21, the relative setting of second heat conduction face 22, the heat conductivity of second heat conduction face 22 is greater than first heat conduction face 21, and the heat dispersion of second heat conduction face 22 is greater than first heat conduction face 21, and two heat conduction faces are transparent material, are convenient for observe the particle that receives a little, and consequently, the temperature of second heat conduction face 22 is less than the temperature of first heat conduction face 21, also makes the temperature of the particle fluid that receives a little less than the temperature of second heat conduction face 22. The sample chamber 24 further includes a gasket 23 for sealing the sample chamber 24, and it can be understood by those skilled in the art that the two heat conducting surfaces 21 may be oppositely disposed or adjacently disposed, or disposed at a predetermined included angle therebetween, and only the micro-nano particles are driven to move and accumulate along a certain direction. The fluid of this embodiment may be a liquid, such as water, or a mixed liquid of water, or may be a gas, such as a heated gas or a natural gas, and it is only necessary to be able to load micro-nano particles and to allow the micro-nano particles to move freely in the fluid, so as to accumulate by using a thermophoresis effect. Meanwhile, the first heat-conducting surface 21 and the second heat-conducting surface 22 are transparent, can sequentially pass through the first heat-conducting surface and the second heat-conducting surface through infrared rays and bring heat into the fluid, and can also generate heat through electromagnetic radiation so as to enable the sample chamber to generate temperature difference.
In a preferred embodiment, the first heat-conducting surface 21 is made of glass, polymethyl methacrylate (PMMA), Polydimethylsiloxane (PDMS), sapphire, or the like, and the second heat-conducting surface 22 is made of sapphire or diamond having good heat conductivity. The laser irradiates the first heat conduction surface 21, the sample chamber 24 loaded with micro-nano particles and the second heat conduction surface 22 in sequence, and a low-temperature region is generated on the second heat conduction surface 22. Adjusting the laser focus into a sample chamber 24, enabling sample liquid in a laser passing area in the sample chamber 24 to absorb laser and increase the temperature, enabling micro-nano particles in the sample to migrate to a second heat conduction surface 22 with lower temperature through a thermophoresis effect, and meanwhile, generating buoyancy due to the fact that the sample liquid expands when heated so as to generate convection in the sample chamber; in the low-temperature direction near the second heat conduction surface 22, the convection direction points to the laser irradiation point from the periphery, and the conveyor belt is used for converging the surrounding micro-nano particles in the area of the second heat conduction surface 22 of the sample chamber below the laser irradiation point, so that the micro-nano particle accumulation is enhanced.
In one embodiment of the present invention, the micro-nano particles are selected as exosomes, which are membrane vesicles secreted by cells for intercellular communication, and since the exosomes contain proteins and genetic materials related to mother cells, the exosomes are gradually an emerging biomarker for non-invasive tumor diagnosis in recent years. The specific principle of this embodiment based on exosomes is as follows.
Exosome thermophoresis movement model:
vT＝-STD▽T (1)
wherein v isTFor thermophoretic velocity, STAnd D is a Soret coefficient, D is a diffusion coefficient, T is a temperature gradient, and the minus sign at the right end of the model formula indicates that the thermophoresis direction is a low-temperature direction.
The Soret coefficient calculation formula in the above formula (1):
wherein A is the exosome surface area, k is the Boltzmann constant, T is the temperature, shydIs entropy of hydration, beta is coefficient, sigmaeffIs the equivalent charge density of the surface of the exosome, lambdaDHIs the Debye length,. epsilon0ε is the relative permittivity of the vacuum. By combining the above equations (1) to (2), it can be seen that the force of exosome thermophoresis is proportional to the diameter square.
Exosome migration model in thermal convection:
Res＝ρa|u-Vp|/η (5)
wherein, VpThe moving speed of the exosome under the action of thermal convection, a is the diameter of the exosome, u is the thermal convection speed, CDIs a viscosity coefficient, and can be calculated according to the formula (4) wherein a1、a2、a3Is constant, ResReynolds number for the relative motion can be calculated according to equation (5), g being the acceleration of gravity, ρpIs the average density of exosomes, ρ is the sample liquid density. By combining equations (3) to (5), it can be seen that the viscous resistance of the exosome to heat convection is proportional to the diameter.
Comparing the thermophoretic force with the thermal convection viscosity resistance, it can be seen that the larger the object is, the more dominant the thermophoretic force is, the more prone the thermophoretic force is to gather on the bottom surface of the sample chamber, i.e. the second heat-conducting surface in the device of the present embodiment; the smaller the object, the more dominant the viscous resistance to thermal convection, the more likely it is to follow thermal convection without collecting. Therefore, the invention enables the micro-nano particles in the liquid to flow and gather through the thermophoresis effect. Of course, the above model is applicable to all micro-nano particles, and the particle size of the micro-nano particles is not limited.
Continuing to refer to fig. 1, the signal amplifying unit 3 includes a microscope disposed in the micro-nano particle accumulation region of the sample chamber unit 2, and includes an objective lens 31 aligned with the accumulated micro-nano particles, a reflector 32, and an observation light source 33, so that the micro-nano particles can be observed more clearly through the microscope. The signal acquisition unit 4 is a CCD camera, and may be any instrument capable of detecting optical signals, and photographs the micro-nano particles through a microscope to acquire information.
In this embodiment, for exosome signal detection, an exosome sample is incubated with a fluorescence-labeled aptamer, so that the fluorescence-labeled aptamer is specifically bound to a target protein on the surface of an exosome, and the exosome is labeled with fluorescence; putting the incubated exosome sample into a sample chamber 24, introducing thermophoresis effect and convection through laser heating, and amplifying a fluorescence signal marked on the exosome in the sample chamber; recording fluorescence signals before and after laser irradiation through a CCD (charge coupled device), and analyzing the fluorescence signals before and after laser irradiation to obtain the abundance of the exosome surface target protein; by using a series of aptamers capable of binding different target proteins, the protein map of the surface of the exosome can be obtained, and corresponding index parameters of the exosome can be finally determined through analysis.
In this embodiment, the method for detecting micro-nano particles includes:
step a, heating a micro-nano particle sample in a sample chamber unit 2 from one side, and generating a thermophoresis effect in the sample chamber unit 2 so as to converge micro-nano particles on one low-temperature side in the sample chamber unit 2;
in the step a, the sample fluid is heated and expanded to generate buoyancy, so that convection is generated in the sample chamber unit 2, the convection direction points to the heating area of the sample chamber unit 2 from the periphery in the low-temperature area of the sample chamber unit 2, and the surrounding micro-nano particles are converged on the low-temperature side of the sample chamber unit 2.
And b, collecting corresponding index information of the micro-nano particles and analyzing corresponding indexes through the micro-nano particles accumulated on the low-temperature side in the sample bin unit 2.
Specifically, the present embodiment performs signal detection on exosomes, and as shown in fig. 2, the process is as follows:
step a1, incubating an exosome sample with a fluorescence-labeled aptamer, and specifically binding the fluorescence-labeled aptamer with a target protein on the surface of an exosome, so as to label the exosome with fluorescence;
step a2, putting the incubated exosome sample into a sample chamber, introducing thermophoresis effect and convection through laser heating, and gathering exosomes on one side of the sample chamber at low temperature so as to amplify the fluorescence signal marked on the exosomes in the sample chamber;
step a3, acquiring fluorescence signals before and after light irradiation, and analyzing the fluorescence signals before and after laser irradiation to obtain the abundance of the exosome surface target protein;
step a4, using a series of aptamers that bind to different target proteins, an exosome surface protein map is derived.
The following describes the micro-nano particle detection system and method by specific embodiments.
Example 1
Incubating an exosome sample with a fluorescence-labeled aptamer, wherein the selected aptamer is an oligonucleotide fragment which is screened out by an in vitro screening technology SELEX (exponential enrichment ligand system evolution) and can specifically bind to proteins or other small molecular substances, specifically, the fluorescence-labeled aptamer is single-stranded DNA (deoxyribonucleic acid) with 40 bases, the diameter of a coil in a sample liquid is less than 5 nanometers, and the diameter of the exosome is 30-150 nanometers; aptamers specifically recognizing CD63 protein were applied to exosomes in the supernatant of a375 cell (human melanoma cell) culture medium. The end of the aptamer can be modified with a fluorophore by standard means, and the aptamer labels the fluorescence carried by the aptamer when the aptamer interacts specifically with the target protein on the surface of the exosome. The exosome sample of this example was cell culture medium supernatant, and the incubation conditions of the sample were: 2 hours incubation time, aptamer concentration 0.1 micromole per liter, incubation temperature room temperature.
Wherein, the laser adopts 1480nm wavelength infrared laser for sample heating, the power is 200 milliwatts, and the laser spot diameter of the focus is about 200 microns. As the sample liquid generally comprises water as the main component, and the water has an absorption peak near the 1480nm band, those skilled in the art can understand that the heating mode is not limited to laser irradiation, the wavelength is not limited to 1480nm, the laser irradiation direction is not limited to top-down irradiation, and the power is selected depending on the irradiation direction, the spot diameter, the wavelength and other factors, and is not limited to 200 mW. In this embodiment, the laser is irradiated from top to bottom, the upper heat conduction surface of the sample chamber is made of a transparent material, such as glass, PMMA, PDMS, and the lower heat conduction surface is made of sapphire with better heat conductivity, and a low-temperature region is formed on the bottom surface to collect the exosome thermophoresis. The thickness of going up the heat conduction face is 1mm, and the thickness of lower heat conduction face is 1mm, and the height of intermediate shim and sample room is 240 mm.
Operating according to the exosome-based signal detection method, when the aptamer recognizes and combines with the exosome surface protein, the fluorescent label on the aptamer is gathered in the bottom area of the sample chamber below the laser light spot along with the exosome, and an enhanced fluorescent signal is generated; when the aptamer does not recognize the surface protein of the exosome, the free aptamer cannot converge due to small size, and the signal is not enhanced. In the present example, as shown in fig. 3, the CD63 protein is widely present on the surface of exosomes of various cells, and after laser irradiation, a distinct fluorescence signal appears, indicating that the surface of exosomes of a375 cells has CD63 protein.
And exciting and receiving a fluorescence signal labeled on the aptamer combined with the exosome by using a fluorescence microscope, wherein the wavelength of exciting and receiving fluorescence is related to the characteristic of the labeled fluorescent luminescent group, in the embodiment, the excitation/emission wavelength of the luminescent group Cy5 is 649/666nm, and the fluorescence signal is recorded by a CCD (charge coupled device) connected with the fluorescence microscope. And recording fluorescence signals before and after laser irradiation through the CCD, and analyzing the fluorescence signals before and after laser irradiation to obtain the abundance of the exosome surface target protein.
Example 2
This example, using serum samples from cervical cancer patients, the abundance of exosome 7 surface proteins (CD63, PTK7, EpCAM, HepG2, HER2, PSA, CA125) in serum samples was measured using 7 different aptamers and compared to healthy human serum samples.
The adopted exosome operation method, the laser, the sample chamber, the microscope and the CCD camera are the same.
As shown in the figure 4, the serum exosome of the cervical cancer patient is known to highly express the CD63 protein, and cancer-related markers PTK7, EpCAM, HepG2, HER2, PSA and CA125, wherein the CA125 can be used as a traditional cervical cancer marker, and a part of cervical cancer patients also have high HER2 expression. Tumor markers PTK7 and EpCAM are generally considered to be associated with various cancers, HepG2 is mainly specific for liver cancer, and PSA is mainly specific for prostate cancer. However, these tumor markers do not have a strict correlation with a certain cancer. However, in the process of tumor growth or cancer metastasis to other organs, after multiple division and proliferation, the cells continuously generate gene mutation, and show changes in molecular biology or genes, so that the tumor markers do not have a strict corresponding correlation relationship with a certain cancer. In the embodiment, PTK7, EpCAM and HepG2 are detected in the serum of the cervical cancer patient, so that the potential of the method in capturing gene mutation or metastasis of tumors is shown. In addition, the expression of CD63 as a protein universally expressed by an exosome in cancer patients is higher than that of healthy people, and the result is consistent with the result obtained by the existing traditional detection method.
The method was further applied to a large number of true 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 cases of healthy persons. As shown in FIG. 5, the method can detect the differences of the expression levels of various proteins in the serum exosomes of various cancer patients and healthy people. The difference of the expression amount of the serum exosome protein among different types of cancers is mainly shown in that HER2 is highly expressed in breast cancer and cervical cancer, CA125 is highly expressed in ovarian cancer and cervical cancer, PSA is not expressed in the types of the detected cancers, and EpCAM, PTK7 and CD63 are highly expressed in various cancers. These results are all consistent with the detection results of the existing methods.
The method can sensitively detect the difference of the expression levels of the exosome surface protein, including the cancer marker, in the serum of the cancer patient and the serum of the healthy person. And the detection method using the exosome as the cancer tumor marker is more convenient, sensitive and effective: the tumor markers for traditional cancer screening or physical examination are limited in types (limited by the available expensive antibodies and reagents) and are not so sensitive as to result in false negatives, i.e., the markers are not detected by the patient, for example, the result of CA125 expression in the venous blood test report of the cervical cancer patient in this embodiment is within the normal range. The method does not need expensive antibodies, and can use aptamers capable of being specifically combined with the protein of the corresponding tumor marker according to the detection requirement.
Example 3
In this embodiment, the micro-nano particles used are non-biological micro-nano particles, specifically fluorescent polystyrene microspheres, which are manufactured by a thermo dissher brand, have a diameter of 50 to 200 nanometers and a mass fraction of 0.001%, and are dissolved in an aqueous solution containing 0.02% of Tween 20. The laser, sample chamber and microscope and CCD camera are the same as in examples 1 and 2 above.
As shown in fig. 5, all the fluorescent microspheres with different diameters are highly converged at the laser spot, and the visible convergence degree and the fluorescence intensity of the fluorescent image according to the fluorescence measurement gray value and the fluorescent image are enhanced along with the increase of the particle diameter, which is consistent with the working principle of this embodiment, that is, the large particles tend to converge more. This example illustrates that micro-nano particles, whether biological or non-biological, are all applicable to the concept of the present technical solution.
Example 4
The micro-nano particles are free protein, nucleic acid and other biological macromolecules or protein, nucleic acid and other biological macromolecules of which exosomes are not exposed on the surface, and the free biological macromolecules cannot be directly accumulated by adopting the thermophoresis effect of each embodiment, so that the mechanism of the embodiment is that an antibody or an aptamer capable of being specifically combined with target protein and nucleic acid is modified on the surface of a micron-sized sphere to obtain an immune microsphere, and the immune microsphere is incubated with a sample containing the target biological macromolecules, combined with the target biological macromolecules and labeled with fluorescence. The microspheres are highly converged through the thermophoresis effect, so that the fluorescence signal of the target biomacromolecule is highly amplified, and the abundance of the target biomacromolecule is detected through the intensity of fluorescence.
The particle detection method based on the microsphere carrier comprises the following steps:
step a11, preparing immune microspheres, and incubating the microspheres and antibodies or aptamers together to fix the antibodies or aptamers on the surfaces of the microspheres to obtain the immune microspheres; in this process, excess antibody or aptamer that is not bound to the microspheres is washed away; in this example, polystyrene microspheres were used as the microspheres.
Step b11, incubating the immune microspheres and the sample to be detected, and specifically binding the target protein or nucleic acid in the sample to be detected with the antibody or aptamer on the immune microspheres so as to fix the target protein or nucleic acid on the immune microspheres;
step c11, combining the immune microsphere combined with the target biomolecule and prepared in the step b11 with an antibody or an aptamer carrying a fluorescent group, and labeling the target biomolecule on the immune microsphere with fluorescence through specific recognition;
step d11, heating the immune microsphere sample combined with the target biomolecule in the sample chamber unit 2 from one side, generating thermophoresis effect in the sample chamber unit 2, so as to gather the immune microsphere combined with the target biomolecule at one side of low temperature in the sample chamber unit 2, and amplifying the signal due to fluorescent label enrichment; in the process, by generating thermophoresis, target biomolecules are captured by the immune microspheres so that the equivalent size is enlarged, the target biomolecules are highly enriched and signals are amplified, and signals with small equivalent sizes but not target biomolecules in a free state cannot be amplified.
Step e11, collecting the corresponding index information of the immune microspheres combined with the target biomolecules and analyzing the corresponding index through the immune microspheres combined with the target biomolecules accumulated on the low temperature side in the sample chamber unit 2. In the process, fluorescent signals before and after light irradiation are obtained, and the abundance of the exosome surface target protein is obtained by analyzing the fluorescent signals before and after laser irradiation; a series of aptamers capable of binding to different target proteins were used to map exosome surface proteins.
In the embodiment, immune microspheres coated with antibodies on the surface are used for capturing free protein markers in whole blood of ovarian cancer patients, infrared laser is used for generating thermophoresis to amplify and detect fluorescent signals of the protein markers, the abundance of the protein markers to be detected is determined, the result accords with the result of a traditional detection method, and molecular information is provided for cancer detection. In this example, for ovarian cancer, EpCAM, CA-125, CA19-9, CD24, HER2, MUC18, EGFR, CLDN3, CD45, CD41, and D2-40 were selected as protein markers, specific antibodies (purchased from abcam) corresponding to these protein markers were prepared as immuno-microspheres, and each antibody was prepared separately as a microsphere specifically for detection of one marker. For the preparation of antibody-coated immune microspheres, reference is made to the standard procedures, which are briefly described here: polystyrene microspheres of 1 micron diameter were incubated with 5. mu.g/ml antibody for 1 hour at room temperature, after which excess unreacted antibody was removed by ultrafiltration. The diameter of the microspheres is not limited to 1 micron, and the microspheres can be converged as long as the size of the microspheres reaches thermophoresis; the material is not limited to polystyrene, and any material can be used as long as the antibody can be successfully attached to the material without affecting the activity of the antibody and the protein marker to be detected, the concentration of the antibody and the incubation temperature and time are not limited to the specific values in the example, and the material is changed by referring to the actual antibody brand batch and the specific experimental conditions.
In this embodiment, 11 kinds of immune microspheres are prepared by the above steps to detect the 11 kinds of markers, 1.1 μ L of patient serum is diluted 100 times and divided into 11 parts, the 11 parts are mixed with the 11 kinds of immune microspheres respectively and incubated at room temperature for 1 hour, the antibody with the fluorescent label and the microspheres capturing the protein markers to be detected are incubated, and the protein markers are fluorescently labeled. And the detection system of each embodiment is adopted for detection. The above procedure was repeated for 10 ovarian cancer patients and 10 healthy persons, and the expression levels of 11 protein markers in 20 serum samples were measured, as shown in FIGS. 7 and 8. Due to cancer heterogeneity, the serum marker expression was not exactly the same for each patient, but was significantly higher in the population than for healthy samples. It is important that each protein marker is used as a single cancer detection standard, and the accuracy is not high. The sum of the expression levels of the proteins in the 11 is used as a detection standard, and the ovarian cancer and the healthy sample can be accurately distinguished by detection. The use of more diagnostically significant markers will greatly improve the diagnostic accuracy, but the cost increases with the number of markers, and antibodies against a particular marker are rare and expensive. According to the detection method, only 1ng of antibody is needed for each marker, the cost is less than 1 yuan, and other expensive reagents are not needed.
So far, the technical solutions of the present invention have been described in connection with the preferred embodiments shown in the drawings, but it is easily understood by those skilled in the art that the scope of the present invention is obviously not limited to these specific embodiments. Equivalent changes or substitutions of related technical features can be made by those skilled in the art without departing from the principle of the invention, and the technical scheme after the changes or substitutions can fall into the protection scope of the invention.
Claims (4)
1. A particle detection system based on a microsphere carrier is characterized by comprising a heating unit and a sample chamber unit, wherein,
the heating unit is used for heating the sample in the sample chamber unit;
the sample bin unit is internally loaded with an exosome sample and an aptamer with a fluorescent label, the aptamer is an oligonucleotide fragment which is screened out by an in vitro screening technology SELEX and can be specifically bound with protein or other small molecular substances, after the sample bin unit is heated by the heating unit, thermophoresis effect and convection are generated in the sample bin unit, the convection direction points to the heating area of the sample bin unit from the periphery in the low-temperature area of the sample bin unit, the exosome is gathered at the lower-temperature side in the sample bin unit, so that when the aptamer in the sample bin is bound with the surface protein of the exosome, the fluorescent label in the aptamer and the exosome are gathered together in the bottom area of the sample bin below a laser light spot to generate an enhanced fluorescent signal for detection;
the system also comprises a signal acquisition unit, wherein the signal acquisition unit acquires the amplified fluorescent signal to obtain the abundance of the aptamer combined with the surface protein of the exosome, and the detection result is obtained by respectively combining various aptamers with different target proteins on the surface of the exosome combined with the aptamer;
the sample chamber unit comprises a closed sample chamber which is used for loading an exosome sample and an aptamer with a fluorescent label and providing a space for generating a thermophoretic effect, and the sample chamber comprises: a second thermally conductive surface for sealing the sample chamber and accumulating the exosome sample and the fluorescently labeled aptamer;
the temperature near the second heat conduction surface is lower than that of the culture medium, so that a temperature difference is generated between the second heat conduction surface and the exosome combined with the aptamer, a thermophoresis effect is generated, and the exosome combined with the aptamer is driven to move directionally to the second heat conduction surface;
the process of detecting exosomes is as follows:
step a1, incubating an exosome sample with a fluorescence-labeled aptamer, and specifically binding the fluorescence-labeled aptamer with a target protein on the surface of an exosome, so as to label the exosome with fluorescence;
step a2, putting the incubated exosome sample into a sample chamber, introducing thermophoresis effect and convection through laser heating, and converging the exosome on the low-temperature side in the sample chamber to amplify the fluorescence signal marked on the exosome in the sample chamber;
step a3, acquiring fluorescence signals before and after light irradiation, and analyzing the fluorescence signals before and after laser irradiation to obtain the abundance of the exosome surface target protein;
step a4, obtaining an exosome surface protein map by using a series of aptamers combining different target proteins;
exosome thermophoresis movement model:
wherein v isTFor thermophoretic velocity, STIs the Soret coefficient, D is the diffusion coefficient,
the negative sign at the right end of the model formula indicates that the thermophoresis direction is a low-temperature direction;
the Soret coefficient calculation formula in the above formula (1):
wherein A is the exosome surface area, k is the Boltzmann constant, T is the temperature, shydIs entropy of hydration, beta is coefficient, sigmaeffIs the equivalent charge density of the surface of the exosome, lambdaDHIs the Debye length,. epsilon0The vacuum dielectric constant, the epsilon relative dielectric constant, and the exosome thermophoresis stress are in direct proportion to the diameter square;
exosome migration model in thermal convection:
Res＝ρa|u-Vp|/η (5)
wherein, VpThe moving speed of the exosome under the action of thermal convection, a is the diameter of the exosome, u is the thermal convection speed, CDIs a viscosity coefficient, and can be calculated according to the formula (4) wherein a1、a2、a3Is constant, ResReynolds number for the relative motion can be calculated according to equation (5), g being the acceleration of gravity, ρpRho is the viscosity resistance of the heated convection of the exosome at the density of the sample liquid, which is the average density of the exosomes, and is directly proportional to the diameter.
2. The system for detecting particles based on the microsphere carrier according to claim 1, wherein the heating unit is a laser arranged outside the sample chamber unit, the laser irradiates the sample chamber unit, and a light beam sequentially passes through the exosome combined with the aptamer and the second heat-conducting surface to generate a thermophoretic effect on a culture medium where the exosome combined with the aptamer is located.
3. The microsphere carrier-based particle detection system of claim 2, wherein the sample chamber further comprises: the first heat conducting surface is used for sealing the sample chamber, and the second heat conducting surface and the first heat conducting surface can both allow light beams to pass through.
4. The microsphere-based particle detection system of claim 3, wherein the second heat conducting surface is transparent and is made of sapphire or diamond;
the first heat conducting surface is any one or combination of glass, polymethyl methacrylate, polydimethylsiloxane and sapphire.
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