CN118222392A - Exosome capturing chip and exosome capturing method - Google Patents
Exosome capturing chip and exosome capturing method Download PDFInfo
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- CN118222392A CN118222392A CN202410408991.0A CN202410408991A CN118222392A CN 118222392 A CN118222392 A CN 118222392A CN 202410408991 A CN202410408991 A CN 202410408991A CN 118222392 A CN118222392 A CN 118222392A
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- 238000000576 coating method Methods 0.000 claims description 8
- AMGQUBHHOARCQH-UHFFFAOYSA-N indium;oxotin Chemical compound [In].[Sn]=O AMGQUBHHOARCQH-UHFFFAOYSA-N 0.000 claims description 8
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Abstract
The invention discloses an exosome capturing chip, which comprises: a conductive base; the micro-fluidic chip is provided with a liquid inlet and a liquid outlet, the bottom of the micro-fluidic chip is provided with a concave cavity, the concave cavity is respectively communicated with the liquid inlet and the liquid outlet, a conductive micro-needle array is arranged in the concave cavity and comprises a conductive substrate and a plurality of conductive micro-needles arranged on the conductive substrate, tips of the conductive micro-needles face the conductive base, and the bottom surface of the micro-fluidic chip is in closed connection with the top surface of the conductive base. The invention also discloses an exosome capturing method. The invention can improve the accuracy and specificity of exosome capture, and the unique design of the conductive microneedle array is utilized to enhance the specific adsorption of exosomes and obviously reduce the risk of nonspecific adsorption; through the microfluidic chip technology, more efficient and accurate exosome separation and purification can be realized, and a more reliable and efficient technical platform is provided for clinical application and biological research of exosomes.
Description
Technical Field
The invention relates to the technical field of exosome capturing, in particular to an exosome capturing chip and method.
Background
Exosomes are nanoscale vesicles secreted by cells, approximately between 30-150nm in diameter. They play an important role in intercellular communication and can reflect the state and function of the cells from which they are derived, and thus exosomes have great potential in the diagnosis and treatment of diseases. The capturing and analysis of the exosomes are important to fully exert the potential of the exosomes in the biomedical field, and the effective capturing technology can improve the purity and the collection efficiency of the exosomes, so that the accuracy and the reliability of the subsequent analysis are improved. However, due to the small size of exosomes and low concentrations in biological fluids, accurately capturing and isolating exosomes is a challenging task.
Currently available exosome capturing methods include ultracentrifugation, density gradient centrifugation, ultrafiltration, etc., wherein the most common method is ultracentrifugation, which is based on the separation of different protein molecules and vesicles according to the difference in sedimentation rate in a homogeneous suspension. The method has the following defects: 1) A great deal of time is required for waiting for the sedimentation of molecules, the time consumption of the process is long, and the efficiency is low; 2) The recovery rate is unstable, and the capture purity is low; 3) The equipment cost is relatively expensive. The density gradient centrifugation method is a density gradient of a medium, so that cells are layered and separated, exosomes are separated from mixed substances, the method has complicated preparation work in the early stage, the operation is complicated and long-time consuming, the number of the obtained exosomes is not large, and subcellular water loss possibly occurs due to high permeability of a gradient solution, so that the biological functions of the exosomes are affected. The ultrafiltration method is to select ultrafiltration membranes with corresponding molecular weight cut-off according to the size of exosomes, and separate by ultrafiltration technology. The method is simple to operate, and has higher yield and efficiency, but non-exosome components smaller than the membrane holes or hybrid proteins with good flexibility can pass through the filter membrane, so that the exosome purity is insufficient, and in addition, the exosome can block the membrane holes to influence the separation efficiency.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide an exosome capturing chip and a exosome capturing method.
In order to achieve the above object, an embodiment of the present invention provides the following technical solution:
an exosome capture chip comprising:
a conductive base;
The micro-fluidic chip is provided with a liquid inlet and a liquid outlet, the bottom of the micro-fluidic chip is provided with a concave cavity, the concave cavity is respectively communicated with the liquid inlet and the liquid outlet, a conductive micro-needle array is arranged in the concave cavity and comprises a conductive substrate and a plurality of conductive micro-needles arranged on the conductive substrate, the tips of the conductive micro-needles face the conductive base, and the bottom surface of the micro-fluidic chip is in closed connection with the top surface of the conductive base.
As a further improvement of the present invention, the conductive base is in contact with a top wall of the cavity, and the conductive micro-needles are conical.
As a further improvement of the invention, the length and the width of the conductive substrate are 22mm and the height is 100 mu m, and the round diameter of the bottom surface of the conductive micro needle is 200 mu m and the height is 200 mu m.
As a further improvement of the invention, a groove is arranged at the bottom of the microfluidic chip, one side wall of the groove is communicated with the concave cavity, the other side wall of the groove penetrates through the microfluidic chip, a conductive plate outwards extends from the outer side of the conductive substrate, and the conductive plate is matched with the groove.
As a further improvement of the present invention, the conductive plate is L-shaped.
As a further improvement of the invention, the length of the micro-fluidic chip is 50mm, the width is 40mm and the height is 3.4mm, and the length of the concave cavity is 42mm, the width is 28mm and the height is 400 μm.
As a further improvement of the invention, the conductive base comprises a glass plate, and an indium tin oxide coating layer arranged on the glass plate.
As a further improvement of the invention, the conductive microneedle array is formed by casting PLA material through a template and then magnetron sputtering.
The exosome capturing method is characterized by using the exosome capturing chip and comprises the following steps:
(1) Exosome sample preparation: collecting an exosome sample to be detected, and carrying out proper pretreatment;
(2) Induced charge electroosmosis technique treatment: mixing the exosome sample with electrolyte solution required by induced charge electroosmosis technology, and adjusting proper parameters to induce and gather exosome;
(3) Exosome capture: capturing exosomes near the conductive microneedles by a conductive microneedle array in combination with an induced charge electroosmosis technique;
(4) Washing and purifying: the exosomes are washed and purified using appropriate buffers to remove non-captured impurities and other cellular structures.
As a further improvement of the invention, in the step (2), the electrolyte solution is potassium chloride solution, and the distribution conductivity is 1 mS/m-10 mS/m; the parameters include: the voltage amplitude is 5-20V, and the frequency of the electric signal is 50-500 Hz.
The beneficial effects of the invention are as follows:
(1) Compared with the traditional capturing mode, the method directly guides the exosomes to the vicinity of the conductive micro-needles in a physical mode, reduces the loss caused by multiple treatments and transfers, and improves the capturing efficiency.
(2) The invention can accurately control the behavior of exosomes based on the induced charge electroosmosis technology, so that the exosomes are more concentrated near the conductive micro-needles, and the efficient static capture of exosomes is realized, thereby improving the capture specificity. Meanwhile, compared with the traditional ultracentrifugation method, density gradient centrifugation method and the like which need complex pretreatment steps, the method is simpler and more convenient to operate, and the experiment time and cost are reduced.
(3) The method solves the problems that the traditional capturing technology relies on modes such as antibody labeling, magnetic attraction and the like, reduces the complexity of operation and the capturing difficulty, and improves the capturing success rate and the raw material utilization rate.
(4) The invention utilizes the unique design of the conductive microneedle array, enhances the specific adsorption of exosomes, obviously reduces the risk of nonspecific adsorption, improves the accuracy and specificity of exosome capture, effectively overcomes the limitations existing in the traditional mode, can realize more efficient and accurate separation and purification of nanoscale cell structures of exosomes and the like, provides a more reliable and efficient technical platform for clinical application and biological research of exosomes, and has important significance for researching cell structures, performing biological analysis and monitoring and the like.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments described in the present invention, and other drawings may be obtained according to the drawings without inventive effort to those skilled in the art.
FIG. 1 is a schematic overall structure of a preferred embodiment of the present invention;
FIG. 2 is a schematic exploded view of a preferred embodiment of the present invention;
FIG. 3 is a side view of a preferred embodiment of the present invention;
FIG. 4 is an enlarged view of a portion of a conductive microneedle array according to a preferred embodiment of the present invention disposed in a cavity;
FIG. 5 is a top view of a preferred embodiment of the present invention;
Fig. 6 is a bottom view of a microfluidic chip-mounting cavity according to a preferred embodiment of the present invention;
FIG. 7 is a cross-sectional view of FIG. 6;
FIG. 8 is a bottom perspective view of a conductive microneedle array according to a preferred embodiment of the present invention;
FIG. 9 is a top view of FIG. 8;
In the figure: 1. the micro-fluidic chip comprises a conductive base, 11, a glass plate, 2, a micro-fluidic chip, 21, a liquid inlet, 22, a liquid outlet, 23, a concave cavity, 231, a top wall, 24, a groove, 3, a conductive micro-needle array, 31, a conductive substrate, 311, a conductive plate, 32 and conductive micro-needles.
Detailed Description
In order to make the technical solution of the present invention better understood by those skilled in the art, the technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, shall fall within the scope of the present invention.
Referring to fig. 1-5, an exosome capturing chip is disclosed in an embodiment of the present application, including: a conductive base 1; the micro-fluidic chip 2, the micro-fluidic chip 2 is provided with a liquid inlet 21 and a liquid outlet 22, the bottom of the micro-fluidic chip 2 is provided with a concave cavity 23, the concave cavity 23 is respectively communicated with the liquid inlet 21 and the liquid outlet 22, a conductive micro-needle array 3 is arranged in the concave cavity 23, the conductive micro-needle array 3 comprises a conductive substrate 31 and a plurality of conductive micro-needles 32 arranged on the conductive substrate 31 in an array manner, the tip parts of the conductive micro-needles 32 face the conductive base 1, and the bottom surface of the micro-fluidic chip 2 is in closed connection with the top surface of the conductive base 1.
The arrangement of the liquid inlet 21, the liquid outlet 22 facilitates the flow of liquid into the cavity 23 and out of the cavity 23. When the conductive microneedle array 3 is assembled in the cavity 23, a gap exists between the inner side wall of the cavity 23 and the outer side of the conductive substrate 31, so that the circulation of liquid is facilitated. The bottom surface of the micro-fluidic chip 2 is in closed connection with the top surface of the conductive base 1, so that the concave cavity 23 is a closed space, and leakage of liquid is avoided. A local electric field is formed near the conductive microneedle 32, and the local electric field causes fluid to swirl, so that the fluid swirl captures the exosomes, and the exosomes are captured in the swirl.
Preferably, the conductive base 1 comprises a glass plate 11, an indium tin oxide coating (not shown) provided on the glass plate 11. The glass plate 11 has chemical stability, is not easy to react with chemical substances in the microfluidic chip 2, and is convenient for keeping the purity of a sample and avoiding chemical pollution. The conductivity of the conductive mount 1 is ensured by providing an indium tin oxide coating.
Preferably, the glass plate 11 has a length L1 of 64mm, a width W1 of 60mm, a height H1 of 1.5mm, and a thickness of the indium tin oxide coating of 0.5 μm. The indium tin oxide coating is not shown in the figures because of its small thickness.
Preferably, the conductive base 31 contacts the top wall 231 of the cavity 23 and the conductive micropins 32 are conical. The processing of the conductive micro-needle 32 is facilitated, and the local electric field intensity formed at the conical conductive micro-needle 32 is convenient for capturing the exosome.
Referring to fig. 3-5, the conductive substrate 31 has a length L2 and a width W2 of 22mm, a height H2 of 100 μm, a bottom circular diameter Φ1 of the conductive micro-needles 32 of 200 μm, and a height H3 of 200 μm. In order to facilitate the orderly arrangement of the plurality of conductive micro-needles 32, it is preferable that the distance D1 between the adjacent conductive micro-needles 32 is 200 μm. Preferably, the conductive microneedle array 3 is composed of thousands of fine conical conductive microneedles 32 arranged in order on the conductive substrate 31. The conductive micro-needle array 3 is formed by casting PLA material through a template and then magnetron sputtering, and a layer of conductive gold film is covered on the surface through magnetron sputtering, so that good conductivity is achieved, and the conductive substrate 31 and the plurality of conductive micro-needles 32 are obtained, so that the conductive micro-needle array 3 is formed. The concrete process of template pouring comprises the following steps: placing polylactic acid, namely PLA solid particles, on a prepared PDMS template at room temperature, placing the template into a vacuum high-temperature heating box, adjusting the heating temperature to 200 ℃ to ensure that the PLA particles are melted and kept for 20 minutes, taking out the template, pressing the melted PLA particles on the PDMS template by using a glass sheet, removing the glass sheet under the condition of the room temperature of an experiment, taking out the PLA microneedle array by using tweezers, placing aside to wait for a modification step, and radiating ultraviolet to improve the hydrophilicity of the PLA microneedle array.
PLA microneedle array conductivity process: polylactic acid, namely PLA, is used as a common polymer material and is not conductive, a magnetron sputtering method is needed to be adopted for conducting the PLA microneedle array, a layer of gold is sputtered on the surface of the microneedle by using a magnetron sputtering coating instrument, the gold is attached to the surface of the PLA microneedle array, metal chromium is doped into the metal gold, the bonding capability of PLA and gold is improved due to the existence of the metal chromium, and the microneedle can conduct after a conducting layer is deposited on the surface, so that the conductive microneedle array 3 is obtained.
Referring to fig. 6-9, a groove 24 is formed at the bottom of the microfluidic chip 2, one side wall of the groove 24 is communicated with the cavity 23, the other side wall of the groove 24 penetrates through the microfluidic chip 2, a conductive plate 311 extends outwards from the side of the conductive substrate 31, and the conductive plate 311 is matched with the groove 24. External wires are convenient to connect through the conducting plate 311 to form the electric field, in addition, through conducting plate 311 chucking cooperation in recess 24, avoid liquid to follow this recess 24 seepage, can confirm the position of electrically conductive microneedle array 3 in cavity 23 simultaneously fast, and realize the spacing to electrically conductive microneedle array 3 through the spacing of conducting plate 311, further improve and catch stability. Specifically, the conductive plate 311, the conductive substrate 31 and the conductive micro-needles 32 are integrally processed and manufactured into a whole through template casting, and the whole is subjected to magnetron sputtering of a gold layer.
Preferably, the conductive plate 311 has an L shape.
Referring to fig. 1, the microfluidic chip 2 has a length L3 of 50mm, a width W3 of 40mm, a height H4 of 3.4mm, a length L4 of 42mm, a width W4 of 28mm, and a height H5 of 400 μm. The distance D2 of the microfluidic chip 2 from the long side of the glass plate 11 was 10mm, and the distance D3 of the microfluidic chip 2 from the wide side of the glass plate 11 was 7mm. The recess 23 is spaced apart from the long side of the microfluidic chip 2 by a distance D4 of 6mm.
By bonding the conductive mount 1 with the microfluidic chip 2: the conductive base 1 is cleaned by deionized water, the surface of the micro-fluidic chip 2 made of PDMS is cleaned by using an adhesive tape to remove dust, then the conductive base 1 and the micro-fluidic chip 2 are placed into a plasma machine, the bonding surfaces of the conductive base 1 and the micro-fluidic chip 2 are guaranteed to be subjected to plasma treatment upwards, the power of the plasma machine is set to be 10W, an oxygen switch is set to be 8-10NI/h, and the treatment is carried out for 32s. Taking out the conductive base 1 and the microfluidic chip 2, and dripping 20 mu L of deionized water on the glass surface with the electrode structure, so that the microfluidic chip 2 can be conveniently moved and aligned on the glass surface, and the electrode structure of the embodiment is an indium tin oxide coating; then placing one surface of the microfluidic chip 2 with the concave cavity 23 on the wet glass surface, observing under a microscope, aligning the concave cavity 23 with the electrode structure according to a preset scheme, and standing for 10 minutes to enable the two surfaces to be attached together; and finally transferring the chip onto a hot plate, lightly pressing the chip on the surface by using weights, and placing the chip for 2 hours at the temperature of 80 ℃ to enable the conductive base 1 to be connected with the microfluidic chip 2 in a bonding way, thus obtaining the bonded capture chip.
To facilitate the liquid flow into and out of the cavity 23, it is preferred that the diameter Φ2 of the liquid inlet 21 and the diameter Φ3 of the liquid outlet 22 are both 2.4mm.
The invention is based on induced charge electroosmosis technology and conductive microneedle technology, an electric field is applied in a system filled with electrolyte, and conductive plates 311 and indium tin oxide coatings are respectively externally connected with wires to be connected with a power supply to form an electric field, so that free electrons in a suspension electrode are induced to migrate to form dipole moment and conductive current. After the stable state is formed, the positive and negative electric layers can move along different directions of the electric field lines due to the electric double layers with opposite polarities on the surfaces of the suspension electrodes, so that convection effect, namely ICEO vortex, is formed in the cavity 23 space, particles are gathered, and the gathering state is stable by adjusting the amplitude and the frequency of alternating current signals. Under the directional control effect of ICEO vortex on micro-flow, the aggregated exosomes can be enriched on a plurality of conductive micro-needles 32 distributed in an array, so that the exosomes can be rapidly and efficiently captured. The method can realize the efficient capture of cell structures such as exosomes by inducing a charge electroosmosis technology, solves the problems that the traditional capture technology relies on modes such as antibody labeling, magnetic attraction and the like, reduces the complexity of operation and the capture difficulty, and improves the capture success rate and the raw material utilization rate.
The embodiment of the invention also provides an exosome capturing method, and the exosome capturing chip adopting the embodiment is provided. In order to better illustrate the method of using the exosome capture chip of the present invention, the exosome capture method of the present invention is described in detail below with reference to specific examples.
An exosome capturing method using the exosome capturing chip of the above embodiment includes the following steps:
(1) Exosome sample preparation: the exosome sample to be tested is collected and suitably pre-treated.
Specifically, pretreatment involves centrifugation of the exosome sample to remove relatively heavy microparticles and unwanted cell debris.
(2) Induced charge electroosmosis technique treatment: mixing the exosome sample with electrolyte solution required by induced charge electroosmosis technology, and adjusting proper parameters to induce and aggregate exosome.
Specifically, the electrolyte solution is potassium chloride solution, and the distribution conductivity is 1 mS/m-10 mS/m; the parameters include: the voltage amplitude is 5-20V, and the frequency of the electric signal is 50-500 Hz.
(3) Exosome capture: exosomes are captured in the vicinity of the conductive microneedles 32 by the conductive microneedle array 3 in combination with an induced charge electroosmosis technique.
By the physical actions of the array arrangement of the plurality of conductive micro-needles 32 of the conductive micro-needle array 3 and the shape of the conductive micro-needles 32, a local electric field is formed near the conductive micro-needles 32, and the local electric field can cause fluid vortex flow, so that the fluid vortex captures the exosomes, and the exosomes are captured in the vortex.
(4) Washing and purifying: the exosomes are washed and purified of the non-trapped impurities and other cellular structures by injecting a suitable buffer into the well 23 through the liquid inlet 21.
Specifically, the buffer may be a 1mS/m potassium chloride solution.
The captured exosomes may be subsequently detected and analyzed, e.g. by microscopic observation of morphology, or proteomic analysis using mass spectrometry techniques, or RNA analysis by qPCR or the like.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Furthermore, it should be understood that although the present disclosure describes embodiments, not every embodiment is provided with a separate embodiment, and that this description is provided for clarity only, and that the disclosure is not limited to the embodiments described in detail below, and that the embodiments described in the examples may be combined as appropriate to form other embodiments that will be apparent to those skilled in the art.
Claims (10)
1. An exosome capture chip, comprising:
a conductive base;
The micro-fluidic chip is provided with a liquid inlet and a liquid outlet, the bottom of the micro-fluidic chip is provided with a concave cavity, the concave cavity is respectively communicated with the liquid inlet and the liquid outlet, a conductive micro-needle array is arranged in the concave cavity and comprises a conductive substrate and a plurality of conductive micro-needles arranged on the conductive substrate, the tips of the conductive micro-needles face the conductive base, and the bottom surface of the micro-fluidic chip is in closed connection with the top surface of the conductive base.
2. The exosome trapping die according to claim 1, wherein the conductive substrate is in contact with a top wall of the cavity, and wherein the conductive micro-needles are conical.
3. The exosome capture chip of claim 2, wherein the conductive substrate has a length and a width of 22mm and a height of 100 μm, and the conductive microneedle has a bottom circular diameter of 200 μm and a height of 200 μm.
4. The exosome capturing chip according to claim 1, wherein a groove is formed in the bottom of the microfluidic chip, one side wall of the groove is communicated with the concave cavity, the other side wall of the groove penetrates through the microfluidic chip, and a conductive plate extends outwards from the outer side of the conductive substrate and is matched with the groove.
5. The exosome capture chip of claim 4, wherein the conductive plate is L-shaped.
6. The exosome capture chip of claim 1, wherein the microfluidic chip has a length of 50mm, a width of 40mm, and a height of 3.4mm, and the cavity has a length of 42mm, a width of 28mm, and a height of 400 μm.
7. The exosome capture chip of claim 1, wherein the conductive mount comprises a glass plate, an indium tin oxide coating disposed on the glass plate.
8. The exosome capture chip of claim 1, wherein the conductive microneedle array is formed by casting PLA material through a template and then magnetron sputtering.
9. An exosome capture method using an exosome capture chip according to any one of claims 1-8, comprising the steps of:
(1) Exosome sample preparation: collecting an exosome sample to be detected, and carrying out proper pretreatment;
(2) Induced charge electroosmosis technique treatment: mixing the exosome sample with electrolyte solution required by induced charge electroosmosis technology, and adjusting proper parameters to induce and gather exosome;
(3) Exosome capture: capturing exosomes near the conductive microneedles by a conductive microneedle array in combination with an induced charge electroosmosis technique;
(4) Washing and purifying: the exosomes are washed and purified using appropriate buffers to remove non-captured impurities and other cellular structures.
10. The method of claim 9, wherein in the step (2), the electrolyte solution is a potassium chloride solution, and the distribution conductivity is 1mS/m to 10mS/m; the parameters include: the voltage amplitude is 5-20V, and the frequency of the electric signal is 50-500 Hz.
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