CN113083385B - Capture chip for capturing tumor specific extracellular vesicles and manufacturing method thereof - Google Patents
Capture chip for capturing tumor specific extracellular vesicles and manufacturing method thereof Download PDFInfo
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Abstract
The invention discloses a capturing chip for capturing tumor specific extracellular vesicles, which comprises a plurality of microarrays; each microarray comprises a substrate, and a micro-channel and a filtrate storage tank which are arranged in the substrate; the micro flow channel is provided with a main flow channel and branch flow channels, a feed port and a discharge port are arranged on the branch flow channels corresponding to the substrate, one ends of the branch flow channels are crossed and communicated to the main flow channel, and the other end of the main flow channel is communicated to the filtrate storage tank through a filtrate port; the feeding hole is used for dropwise adding a solution to be detected and a microsphere compound, and the size and the shape of the microsphere compound are consistent; the microsphere compound can slowly move in the branch flow channel and adsorb the tumor specific extracellular vesicles, the solution to be detected is separated to a filtrate storage tank through a filtrate port at the bottom of the main flow channel, and the microsphere compound combined with the tumor extracellular vesicles can be taken out through a discharge port. The capture chip is combined with a microfluidic technology, so that the surface area of a flow channel is increased, the movement resistance of the microsphere compound is increased, and the combination time of the antigen and the antibody of the microsphere compound is prolonged.
Description
Technical Field
The invention relates to the technical field of biomedical detection, in particular to a capture chip for capturing tumor specific extracellular vesicles and a manufacturing method thereof.
Background
Early diagnosis of tumors is of great importance for improving diagnostic and therapeutic methods. If the drug fails to intervene in time today, subsequent treatments are costly and survival rates are not high. Among them, tumor screening is more advantageous than early diagnosis of tumors. The presumptive identification of tumors is performed by testing, examining, or other quick and convenient means for a target population that appears healthy and asymptomatic. Further monitor and intervene the tumor in advance, and eliminate the tumor focus before it is formed. Can save lives and reduce the personal, social and economic costs of tumor treatment. Current conventional tumor screening approaches are often associated with some risks. For example, CT scanning can result in exposure of the patient to low doses of radioactivity. Moreover, the frequent false positive results of these screens have led to overdose of patients by physicians and to failure to clear the actual lesion. The tumor screening or the accurate tumor marker inspection result is particularly important for guiding doctors to take medicines. There are some potential tumor signaling molecules-tumor markers in the urine, blood or other body fluids of the human body. The current means of enrichment have also been late when markers are detected in peripheral blood, where metastasis of the cancer often has already occurred.
Extracellular Vesicles (EVs), a type of microvesicles with a bilayer lipid membrane structure secreted by cells. In recent years, it has been found that extracellular vesicles play an important role in communication between cells. The capture antibody is combined with antigen molecules on the surface of the membrane by utilizing specific immunoreaction, and the capture of extracellular vesicles can be realized.
Therefore, the development of a tumor-specific extracellular vesicle capture chip which is simple to manufacture, convenient to operate and control and low in cost is urgently needed at the present stage, so that the efficient capture of the extracellular vesicles in a sample is realized, and the diagnosis and screening of early tumors are realized.
Disclosure of Invention
The invention overcomes the defects of the prior art and provides a capture chip for capturing tumor specific extracellular vesicles.
In order to achieve the purpose, the invention adopts the technical scheme that: a capture chip for capturing tumor-specific extracellular vesicles, the capture chip comprising a plurality of microarrays;
each microarray comprises a substrate and a micro-channel and a filtrate storage tank arranged in the substrate; the micro flow channel is provided with a main flow channel and branch flow channels, a feed inlet and a discharge outlet are arranged above the branch flow channels corresponding to the substrate, one ends of the branch flow channels are crossed and communicated with the main flow channel, and the other end of the main flow channel is communicated with the filtrate storage tank through a filtrate outlet;
the feeding hole is used for dropwise adding a solution to be detected and a microsphere compound, and the size and the shape of the microsphere compound are consistent; the microsphere compound can slowly move in the branch flow channel and adsorb tumor-specific extracellular vesicles, a solution is separated to the filtrate storage tank through the filtrate port at the bottom of the main flow channel, and the microsphere compound combined with the tumor extracellular vesicles can be taken out through the discharge port.
In a preferred embodiment of the present invention, an external magnetic field for slowing down the moving speed of the microsphere composite is disposed above the substrate of the branch flow channel.
In a preferred embodiment of the present invention, a magnetic block is disposed in the interlayer of the substrate of the branch flow channel, and the magnetic block wraps the branch flow channel.
In a preferred embodiment of the present invention, the branched flow channel has an included angle of 5 ° to 10 ° with the horizontal plane.
In a preferred embodiment of the present invention, the bottom surface of the branched channel in the micro flow channel has a plurality of pits or protrusions, and the size of the pits is much smaller than that of the microsphere composite.
In a preferred embodiment of the present invention, only one microsphere composite can pass through the branched flow channel in the aperture direction.
In a preferred embodiment of the present invention, the microsphere composite takes magnetic beads coated with tumor antibodies as a carrier, and the antibodies in the microsphere composite are used for specific antigen-antibody binding with a solution to be detected.
In a preferred embodiment of the present invention, the test solution comprises plasma, serum, urine, cerebrospinal fluid and saliva.
The invention also provides a manufacturing method of the capture chip for capturing the tumor specific extracellular vesicles, which comprises the following steps:
s1, drawing a shape chart of the micro flow channel of the capture chip by using drawing software, and manufacturing a substrate of a single chip by using a printer;
s2, chemically modifying the micro flow channel in the chip by using an acidic buffer solution, and flushing the flow channel by using a mixed buffer solution and using D-PBS as a base solution to form a main flow channel and a branch flow channel of the micro flow channel.
In a preferred embodiment of the present invention, several single chips prepared in S2 are integrated to form an array of capture chips for simultaneously capturing tumor-specific extracellular vesicles in different solutions.
In a preferred embodiment of the present invention, the size of the branched channel is substantially the same as the size of the microsphere composite.
In a preferred embodiment of the present invention, the recess or the protrusion is a flexible buffer layer, which can minimize cell damage caused by collision during the capturing process.
In a preferred embodiment of the present invention, the external magnetic field may be symmetrically distributed on both sides of the micro flow channel.
In a preferred embodiment of the present invention, the external magnetic field generates a gradient magnetic field along the branch flow channel, and the purpose of controlling the flow of the microsphere composite is achieved by adjusting the gradient direction of the external magnetic field.
The invention solves the defects in the background technology, and has the following beneficial effects: the invention provides a capture chip for capturing tumor-specific extracellular vesicles, wherein a plurality of microarrays are integrated in the capture chip, a microfluidic technology is combined, each microarray can be used for detecting tumor-specific extracellular vesicles from different body fluids or blood of a patient, the manufacturing technology of the capture chip is convenient and rapid, the cost is low, and the possibility is provided for large-scale application of the capture chip.
In the invention, a plurality of crossed branch channels are arranged in the micro-channel, so that the surface area of the channels is increased, each channel is not interfered with each other, and only one microsphere compound can pass through the pore diameter direction of each branch channel, so that the antigen-antibody combination of the microsphere compound and tumor specific extracellular vesicles in the branch channels is increased.
The bottom surface of the branch flow channel in the micro flow channel is provided with a plurality of pits or bulges, so that the branch flow channel forms certain resistance to the solution in the micro flow channel, the advancing speed of the solution is reduced, and sufficient reaction time is provided for the subsequent antigen-antibody combination of the microsphere compound.
In the invention, an external magnetic field is arranged above the substrate, or a magnetic block is arranged in an interlayer of the substrate, the movement state of the microsphere composite is interfered by the magnetic field, the magnetic sphere composite moves directionally under the action of the magnetic field, so that the microsphere composite moves along the branch flow channel, the movement resistance of the microsphere composite can be increased under the action of the magnetic field, and the reaction time of the microsphere composite and a solution is further increased.
The invention is combined with the micro-fluidic technology, cooperates with the micro-fluidic micro-scale fluid regulation and control, and carries out the tumor cell-capture with high affinity and high selectivity.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below, it is obvious that the drawings in the following description are only some embodiments described in the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts;
FIG. 1 is a perspective block diagram of a capture chip of a preferred embodiment of the present invention;
FIG. 2 is a perspective view of a microarray according to a preferred embodiment of the present invention;
in the figure: 1. capturing a chip; 2. a microarray; 3. a substrate; 4. a micro flow channel; 5. a main flow channel; 6. a filtrate holding tank; 7. branch flow channels; 8. a feed inlet; 9. and (4) a discharge port.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those specifically described herein, and therefore the scope of the present invention is not limited by the specific embodiments disclosed below.
In the description of the present application, it is to be understood that the terms "center," "longitudinal," "lateral," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," and the like are used in the orientation or positional relationship indicated in the drawings for convenience in describing the present application and for simplicity in description, and are not intended to indicate or imply that the referenced devices or elements must have a particular orientation, be constructed in a particular orientation, and be operated in a particular manner, and are not to be considered limiting of the scope of the present application. Furthermore, the terms "first," "second," and the like are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first," "second," etc. may explicitly or implicitly include one or more of that feature. In the description of the invention, the meaning of "a plurality" is two or more unless otherwise specified.
In the description of the present application, it is to be noted that, unless otherwise explicitly specified or limited, the terms "mounted," "connected," and "connected" are to be construed broadly, e.g., as meaning either a fixed connection, a removable connection, or an integral connection; 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 meaning of the above terms in the present application can be understood by those of ordinary skill in the art through specific situations.
As shown in fig. 1, it is a three-dimensional structure diagram of a capture chip 1 for capturing tumor-specific extracellular vesicles. The capture chip 1 comprises several microarrays 2. Several microarrays 2 are integrated to form a capture chip 1. All the microarrays 2 can simultaneously detect different solutions to be detected, so that whether different parts of different human bodies contain tumor signal molecules or the number of the tumor signal molecules is detected in a short time; or the solution to be measured in the same sample is measured by dividing into a plurality of parts, so that the accuracy is improved.
As shown in fig. 2, each microarray 2 includes a substrate 3 and a micro flow channel 4 and a filtrate holding tank 6 provided in the substrate 3. The micro flow channel 4 is provided with a main flow channel 5 and branch flow channels 7, a feed inlet 8 and a discharge outlet 9 are arranged above the branch flow channels 7 corresponding to the substrate 3, one ends of the branch flow channels 7 are crossed and communicated to the main flow channel 5, and the other end of the main flow channel 5 is communicated to the filtrate storage tank 6 through a filtrate port; the feed port 8 is used for dripping the solution to be detected and the microsphere compound, and the size and the shape of the microsphere compound are consistent; the microsphere compound can slowly move in the branch flow channel 7 and adsorb the tumor specific extracellular vesicles, the solution is separated to the filtrate storage tank 6 through the filtrate port at the bottom of the main flow channel 5, and the microsphere compound combined with the tumor extracellular vesicles can be taken out through the discharge port 9. In the invention, a plurality of crossed branch flow channels 7 are arranged in the micro flow channel 4, so that the surface area of the flow channels is increased, each flow channel is not interfered with each other, and only one microsphere compound can pass through the pore diameter direction of each branch flow channel 7, so that the antigen-antibody combination of the microsphere compound and the tumor specific extracellular vesicles in the branch flow channels 7 is increased.
It should be noted that, in the microsphere composite of the present invention, the magnetic beads coated with the tumor antibody are used as a carrier, and the antibody in the microsphere composite is used for specific antigen-antibody binding with the solution to be detected.
The branch flow channel 7 and the horizontal plane have an included angle of 5-10 degrees. The bottom surface of the branch flow channel 7 in the micro flow channel 4 is provided with a plurality of pits or bulges, and the size of the pits is far smaller than that of the microsphere compound, so that the branch flow channel 7 forms certain resistance to the solution in the micro flow channel, the advancing speed of the solution is reduced, and sufficient reaction time is provided for the subsequent antigen-antibody combination of the microsphere compound.
It should be noted that, the pits or the bumps in the present invention are a flexible buffer layer, which can minimize cell damage caused by collision during the capturing process, and is beneficial to retain the activity and biological function of the tumor extracellular vesicles.
In the invention, an external magnetic field for slowing down the movement speed of the microsphere compound is arranged above the substrate 3 of the branch flow channel 7; or a magnetic block is arranged in the interlayer of the substrate 3 positioned in the branch flow channel 7 and wraps the branch flow channel 7. The invention is not limited to the above two modes, and the main purpose is to interfere the movement state of the microsphere composite through a magnetic field, the magnetic sphere composite moves directionally under the action of the magnetic field, so that the microsphere composite moves along the branch flow channel 7, and the action of the magnetic field can increase the movement resistance of the microsphere composite and further increase the reaction time of the microsphere composite and the solution.
It should be noted that, the external magnetic field in the invention can be symmetrically distributed on both sides of the micro-channel; or the external magnetic field generates a gradient magnetic field along the branch flow channel direction, and when the gradient direction of the external magnetic field is adjusted to be opposite to the flowing direction of the microsphere compound, the flowing speed of the microsphere compound in the liquid to be detected is reduced under the action of magnetic force; the flow of the microsphere composite is further controlled by adjusting the size of the magnetic field gradient.
The size of the branch flow channel is basically consistent with that of the microsphere compound, the size of the branch flow channel is micron scale, the size difference between the branch flow channel and the microsphere compound selectively increases the collision frequency between the microsphere compound and the branch flow channel, and the affinity effect of the microsphere compound and the tumor-specific extracellular vesicles is selectively increased.
Extracellular vesicles are widely present in cell culture supernatants and various body fluids, and thus the test solution of the present invention includes blood, lymph, urine, semen, milk, plasma, serum, cerebrospinal fluid, feces, pleural effusion, pericardial fluid, lymph, chyme, bile, or cell culture supernatant or any combination thereof.
In the prior art, methods of ultracentrifugation, magnetic bead immunocapture, precipitation or filtration are mostly adopted for tumor extracellular vesicles to carry out early separation on exosomes, wherein the traditional ultracentrifugation needs complicated steps and expensive instruments, which may result in low recovery rate and high cost; although high-purity exosomes can be obtained by an antibody affinity method and a density gradient centrifugation method, exosomes in an intact state cannot be obtained, and physiological functions of the exosomes in the intact state cannot be analyzed. On the other hand, standard methods widely used for exosome quantification, such as enzyme-linked immunosorbent assay (ELISA) and western blot, are limited by large sample requirements and low sensitivity. Therefore, the method is more convenient and rapid for capturing the tumor extracellular vesicles, more accurate in the capturing process and lower in cost.
The invention also provides a manufacturing method of the capture chip 1 for capturing the tumor specific extracellular vesicles, which comprises the following steps:
s1, drawing the shape chart of the micro-channel 4 of the capture chip 1 by using drawing software, and manufacturing a substrate 3 of a single chip by using a printer;
s2, chemically modifying the micro flow channel 4 in the chip by using an acidic buffer solution, and flushing the flow channel by using a mixed buffer solution and D-PBS as a base solution to form a main flow channel 5 and a branch flow channel 7 of the micro flow channel 4.
S3, integrating a plurality of single chips prepared in S2 to form an array of capture chips 1, and simultaneously capturing tumor-specific extracellular vesicles in different solutions.
When the kit is used, solutions to be detected of different types or the same type are respectively dripped from a feeding hole 8 on a branch flow channel 7, after the intermission of 5-10s, the microsphere compound is added into the branch flow channel 7 one by one, and the microsphere compound and tumor specific extracellular vesicles in the solution to be detected carry out antigen-antibody reaction in the branch flow channel 7; the solution is filtered through the filtrate port into the filtrate holding tank 6, the microsphere complex remains above the filtrate port, and the microsphere complex bound to the tumor extracellular vesicles can be removed through the discharge port 9.
In light of the foregoing description of the preferred embodiment of the present invention, it is to be understood that various changes and modifications may be made by one skilled in the art without departing from the spirit and scope of the invention. The technical scope of the present invention is not limited to the content of the specification, and must be determined according to the scope of the claims.
Claims (10)
1. A capture chip for capturing tumor-specific extracellular vesicles, the capture chip comprising a plurality of microarrays;
each microarray comprises a substrate and a micro-channel and a filtrate storage tank arranged in the substrate; the micro flow channel is provided with a main flow channel and branch flow channels, a feed inlet and a discharge outlet are arranged above the branch flow channels corresponding to the substrate, one ends of the branch flow channels are crossed and communicated with the main flow channel, and the other end of the main flow channel is communicated with the filtrate storage tank through a filtrate outlet;
the feeding hole is used for dropwise adding a solution to be detected and a microsphere compound, and the size and the shape of the microsphere compound are consistent; the microsphere compound can slowly move in the branch flow channel and adsorb tumor specific extracellular vesicles, a solution to be detected is separated to the filtrate storage tank through the filtrate port at the bottom of the main flow channel, and the microsphere compound combined with the tumor extracellular vesicles can be taken out through the discharge port.
2. A capture chip for capturing tumor-specific extracellular vesicles according to claim 1, wherein: and an external magnetic field for slowing down the movement speed of the microsphere compound is arranged above the substrate of the branch flow channel.
3. A capture chip for capturing tumor-specific extracellular vesicles according to claim 1, wherein: and a magnetic block is arranged in the interlayer of the substrate of the branch flow channel and wraps the branch flow channel.
4. A capture chip for capturing tumor-specific extracellular vesicles according to claim 1, wherein: the branch flow channel and the horizontal plane form an included angle of 5-10 degrees.
5. A capture chip for capturing tumor-specific extracellular vesicles according to claim 1, wherein: the bottom surface of the branch flow channel in the micro flow channel is provided with a plurality of pits or bulges, and the size of each pit is far smaller than that of the microsphere compound.
6. A capture chip for capturing tumor-specific extracellular vesicles according to claim 1, wherein: the branch flow channel can only pass through one microsphere compound in the aperture direction.
7. A capture chip for capturing tumor-specific extracellular vesicles according to claim 1, wherein: the microsphere compound takes magnetic beads coated by tumor antibodies as carriers, and the antibodies in the microsphere compound are combined with the solution to be detected through specific antigens-antibodies.
8. A capture chip for capturing tumor-specific extracellular vesicles according to claim 1, wherein: the test solution comprises plasma, serum, urine, cerebrospinal fluid and saliva.
9. The method for manufacturing a capture chip for capturing tumor-specific extracellular vesicles according to any one of claims 1 to 8, comprising the steps of:
s1, drawing a shape chart of the micro flow channel of the capture chip by using drawing software, and manufacturing a substrate of a single chip by using a printer;
s2, chemically modifying the micro flow channel in the chip by using an acidic buffer solution, and flushing the flow channel by using a mixed buffer solution and using D-PBS as a base solution to form a main flow channel and a branch flow channel of the micro flow channel.
10. The method of claim 9, wherein the capture chip comprises: several single chips prepared in S2 were assembled to form an array of capture chips for simultaneous capture of tumor-specific extracellular vesicles in different solutions.
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Citations (5)
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| CN103084229A (en) * | 2012-01-16 | 2013-05-08 | 中国科学院深圳先进技术研究院 | Micro-fluidic chip, hemocyte analysis system and hemocyte analysis method |
| CN106520698A (en) * | 2016-01-05 | 2017-03-22 | 苏州汶颢芯片科技有限公司 | Cell non-contact culture method based on micro-fluidic chip |
| CN106975526A (en) * | 2016-01-15 | 2017-07-25 | 中国科学院苏州纳米技术与纳米仿生研究所 | Micro-fluidic chip, its preparation method and situ catalytic and detection method |
| CN107213929A (en) * | 2017-06-06 | 2017-09-29 | 国家纳米科学中心 | A kind of micro-nano particle piece-rate system based on interfacial effect |
| CN110713900A (en) * | 2019-11-05 | 2020-01-21 | 深圳先进技术研究院 | Micro-fluidic chip for separating and capturing single cell and preparation method and application thereof |
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| US7244232B2 (en) * | 2001-03-07 | 2007-07-17 | Biomed Solutions, Llc | Process for identifying cancerous and/or metastatic cells of a living organism |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103084229A (en) * | 2012-01-16 | 2013-05-08 | 中国科学院深圳先进技术研究院 | Micro-fluidic chip, hemocyte analysis system and hemocyte analysis method |
| CN106520698A (en) * | 2016-01-05 | 2017-03-22 | 苏州汶颢芯片科技有限公司 | Cell non-contact culture method based on micro-fluidic chip |
| CN106975526A (en) * | 2016-01-15 | 2017-07-25 | 中国科学院苏州纳米技术与纳米仿生研究所 | Micro-fluidic chip, its preparation method and situ catalytic and detection method |
| CN107213929A (en) * | 2017-06-06 | 2017-09-29 | 国家纳米科学中心 | A kind of micro-nano particle piece-rate system based on interfacial effect |
| CN110713900A (en) * | 2019-11-05 | 2020-01-21 | 深圳先进技术研究院 | Micro-fluidic chip for separating and capturing single cell and preparation method and application thereof |
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