CN111073793B - Centrifugal micro-fluidic chip, manufacturing method and application method thereof - Google Patents
Centrifugal micro-fluidic chip, manufacturing method and application method thereof Download PDFInfo
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- CN111073793B CN111073793B CN201911383875.3A CN201911383875A CN111073793B CN 111073793 B CN111073793 B CN 111073793B CN 201911383875 A CN201911383875 A CN 201911383875A CN 111073793 B CN111073793 B CN 111073793B
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- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/02—Form or structure of the vessel
- C12M23/16—Microfluidic devices; Capillary tubes
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- B—PERFORMING OPERATIONS; TRANSPORTING
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- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
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- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
- C12M35/02—Electrical or electromagnetic means, e.g. for electroporation or for cell fusion
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
- C12M41/36—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of biomass, e.g. colony counters or by turbidity measurements
Abstract
The application discloses a centrifugal microfluidic chip, a manufacturing method and an application method thereof, and relates to the technical field of microfluidics. The centrifugal micro-fluidic chip comprises a bearing substrate, a micro-fluidic substrate, an image acquisition device and a wireless power supply transmitting device; the bearing substrate comprises a plurality of super lens arrays; the micro-fluidic substrate comprises a plurality of cell suspension cavities, the cell suspension cavities and the super-lens arrays are arranged in a one-to-one correspondence mode, and one cell suspension annular electrode array is arranged on the outer side of one super-lens array in a surrounding mode; the cells are sorted by the plurality of cavities on the microfluidic chip, and after the cells are captured, the cells are imaged by the superlens array and the image acquisition unit, so that the centrifugal microfluidic chip has the functions of cell sorting, high-resolution cell imaging and the like. The invention has the advantages that the bearing substrate and the microfluidic substrate are assembled in a bonding way, the manufacturing process is simple, and the superlens is integrated on the microfluidic chip, so that a high-efficiency and high-precision cell operation analysis tool is provided for the research of cell mechanics.
Description
Technical Field
The invention relates to the technical field of microfluidics, in particular to a centrifugal microfluidic chip, a manufacturing method and an application method thereof.
Background
The observation of biological cells in their physiological environment and their corresponding physiological forms is crucial to understanding and studying the relationship between cell forms and pathogenesis of some diseases, and laboratories generally adopt equipment tools such as atomic force microscopes to study related cell mechanics and imaging subjects. However, these devices have problems in that they are high in power consumption, low in efficiency, and low in integration due to the need for a separate sample processing process.
In contrast, microfluidics with miniaturization, integration, and automation are effective means to overcome the problems with the above-mentioned research tools; and there is good dimensional compatibility between microfluidics and cells. Therefore, the microfluidics technology provides a promising approach for studying physiological effects caused by stress and deformation applied to cells through efficient and high-precision cell manipulation analysis.
Centrifugal microfluidic technology has been widely studied in the fields of clinical diagnosis, immunology, protein molecular analysis, and the like. The centrifugal microfluidic technology is a reliable way to realize functions such as cell operation and analysis, but the centrifugal microfluidic chip in the prior art does not have an integrated technology with functions such as cell control and high-resolution cell imaging, and cannot realize cell control and high-resolution cell imaging.
Disclosure of Invention
In view of this, the present invention provides a centrifugal microfluidic chip and a method for manufacturing the same, so as to solve the problems that the centrifugal microfluidic chip in the prior art does not have the functions of cell manipulation, high-resolution cell imaging, and the like, and cannot achieve cell manipulation and high-resolution cell imaging.
In order to achieve the purpose, the invention provides the following technical scheme:
a centrifugal microfluidic chip for cell deposition and cell capture, the centrifugal microfluidic chip comprising:
the device comprises a microfluidic substrate, a bearing substrate, a wireless power supply receiving and image collecting device and a wireless power supply transmitting device which are oppositely arranged;
the surface of the microfluidic substrate facing the bearing substrate is provided with a groove structure, the microfluidic substrate is hermetically connected with the bearing substrate, the groove structure forms a plurality of cavities, the plurality of cavities at least comprise a plurality of cell suspension cavities, and the cell suspension cavities are in one-to-one correspondence with the superlens arrays; the microfluidic substrate comprises a through hole structure which is used for installing, ventilating and leading the microfluid to enter the groove structure; the plurality of cavities and the through hole structure form two parallel functional units;
the surface of the bearing substrate facing the microfluidic substrate is provided with a plurality of super lens arrays and a plurality of cell suspension annular electrode arrays; the cell suspension annular electrode arrays are arranged in one-to-one correspondence with the super lens arrays, one cell suspension annular electrode array is arranged on the outer side of one super lens array in a surrounding manner, and the cell suspension annular electrode arrays are arranged in the cell suspension cavity; the surface of the bearing substrate, which is far away from the microfluidic substrate, is provided with a plurality of image acquisition unit installation cavities and control circuit module installation cavities;
the wireless power supply receiving and image collecting device comprises a plurality of image collecting units, a control circuit module and a wireless power supply receiving module, wherein the image collecting units are connected with the control circuit module, and the control circuit module is connected with the wireless power supply receiving module; and the image acquisition unit is embedded in the image acquisition unit installation cavity, and the control circuit module is embedded in the control circuit module installation cavity.
In one embodiment, the plurality of cavities further includes at least two cell sedimentation cavities, the surface of the support substrate facing the microfluidic substrate is further provided with two sets of cell sorting electrode pairs which are arranged in a central symmetry manner, the two sets of cell sorting electrode pairs are arranged in one-to-one correspondence with the two cell sedimentation cavities, and the cell sorting electrode pairs are arranged in the two cell sedimentation cavities.
In one embodiment, the plurality of chambers further comprises a sample injection chamber, a cell precipitation chamber, and a waste liquid chamber;
the through hole structure comprises a sample inlet hole, a sample inlet cavity vent hole, a waste liquid cavity vent hole and a mounting hole;
the sample inlet hole and the sample injection cavity vent hole are respectively positioned at two sides of the top end of the sample injection cavity, and the sample inlet hole and the sample injection cavity vent hole are both communicated with the sample injection cavity;
the waste liquid cavity vent hole is communicated with the waste liquid cavity;
the sample injection cavity is connected with the cell sedimentation cavity through a micro valve;
the cell precipitation cavity is connected with the waste liquid cavity through the waste liquid cavity micro-channel.
In one embodiment, the sample injection cavity and the waste cavity have a first depth, and the cell sedimentation cavity and the waste cavity microchannel have the same second depth, wherein the first depth is greater than the second depth.
In one embodiment, the image acquisition unit is a CMOS image sensor, and a surface of the CMOS image sensor is coated with a filter layer for filtering stray light entering the CMOS image sensor.
In one embodiment, the superlenses of the superlens array are in a concentric ring topology, the numerical aperture of the superlenses being 0.9.
An application method of a centrifugal microfluidic chip is provided, and the application method comprises the following steps:
providing the centrifugal microfluidic chip, a sample solution and a buffer solution, wherein the sample solution comprises a plurality of cells, and the buffer solution is a PBS buffer solution;
adding a buffer solution into a centrifugal micro-fluidic chip, and rotating the centrifugal micro-fluidic chip according to a first preset rotating speed and a first preset time;
adding the sample liquid into the centrifugal microfluidic chip, and rotating the centrifugal microfluidic chip according to a second preset rotating speed and a second preset time;
and when the cells in the sample liquid enter the cell sedimentation cavity, stopping rotating the centrifugal microfluidic chip, applying a preset voltage to the cell suspension annular electrode array on the bearing substrate, and after the cells in the sample liquid are captured by the cell suspension cavity, acquiring image information of the captured cells by the image acquisition unit.
In one embodiment, the application method of the centrifugal microfluidic chip further includes the following steps: and applying a preset voltage to the cell sorting electrode pair on the bearing substrate while rotating the centrifugal micro-fluidic chip according to a preset rotating speed and preset time.
A manufacturing method of a centrifugal microfluidic chip is used for manufacturing the centrifugal microfluidic chip, and comprises the following steps:
providing a bearing substrate body, a micro-fluidic substrate body, a wireless power supply receiving and image collecting device and a wireless power supply transmitting device;
the bearing substrate body comprises a first surface and a second surface which are oppositely arranged, a plurality of image acquisition unit installation cavities, a wireless power supply receiving module installation cavity and a control circuit module installation cavity are manufactured on the first surface of the bearing substrate body, and a cell suspension annular electrode array lead is manufactured at a preset position; manufacturing two sets of cell sorting electrode pairs on the second surface of the bearing substrate body, and manufacturing a cell suspension annular electrode array pair and a super lens array with a concentric ring topological structure at a preset position to obtain a bearing substrate;
manufacturing two sets of groove structures in one surface of the microfluidic substrate body, and manufacturing a through hole structure in the microfluidic substrate body to obtain a microfluidic substrate;
the wireless power supply receiving and image collecting device comprises a plurality of image collecting units, a control circuit module and a wireless power supply receiving module, wherein the image collecting units are connected with the control circuit module, the control circuit module is connected with the wireless power supply receiving module, the image collecting units are installed in an image collecting unit installation cavity, the wireless power supply receiving module is installed in a wireless power supply receiving module installation cavity, and the wireless power supply transmitting device and the wireless power supply receiving module are correspondingly arranged;
and assembling and sealing the surface of the bearing substrate with the super lens array and the surface of the microfluidic substrate with the groove structure to obtain the centrifugal microfluidic chip.
In an embodiment, the method for manufacturing a centrifugal microfluidic chip further includes the following steps:
manufacturing a cell sorting electrode pair lead at a preset position on the first surface of the bearing substrate body;
and manufacturing two sets of cell sorting electrode pairs on the second surface of the bearing substrate body.
The centrifugal microfluidic chip provided by the invention comprises a bearing substrate, a microfluidic substrate, a wireless power supply receiving and image acquisition device and a wireless power supply transmitting device. The carrier substrate includes a plurality of superlens arrays thereon. The microfluidic substrate comprises a plurality of cell suspension cavities, and the cell suspension cavities and the superlens arrays are arranged in a one-to-one correspondence mode. The cell is sorted by a cell sedimentation cavity on the microfluidic chip, the cell is suspended and focused by a cell suspension annular electrode array, and the cell is imaged by a super lens array and an image acquisition unit, so that the centrifugal microfluidic chip has the functions of cell sorting, high-resolution cell imaging 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 used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
Fig. 1 is a schematic structural diagram of a centrifugal microfluidic chip according to an embodiment of the present invention;
FIG. 2 is a schematic diagram of an exploded structure of the centrifugal microfluidic chip shown in FIG. 1;
FIG. 3 is a top view of a microfluidic substrate;
FIG. 4 is a bottom view of a carrier substrate;
FIG. 5 is a top view of a carrier substrate;
FIG. 6 is a bottom view of a cell suspension electroosmosis electrode and concentric ring superlens on a carrier substrate;
FIG. 7 is a bottom view of a superlens on a carrier substrate;
FIG. 8 is a cross-sectional view of a superlens on a carrier substrate;
FIG. 9 is a flow chart of a manufacturing process of a centrifugal microfluidic chip according to an embodiment of the present invention;
FIGS. 10A-10D are process diagrams of fabricating a superlens array according to an embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, 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. It is to be understood that the embodiments described are only a few embodiments of the present invention, and not all embodiments. The components of embodiments of the present invention generally described and illustrated in the figures herein may be arranged and designed in a wide variety of different configurations.
Thus, the following detailed description of the embodiments of the present invention, presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. 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 description of the present invention, it should be understood that the relation indicating the orientation or position such as "above" is based on the orientation or position relation shown in the drawings, or the orientation or position relation which the product of the present invention is usually put into use, or the orientation or position relation which is usually understood by those skilled in the art, and is only for convenience of describing the present invention and simplifying the description, but does not indicate or imply that the device or element to be referred to must have a specific orientation, be constructed and operated in a specific orientation, and thus, cannot be construed as limiting the present invention.
Furthermore, the terms "first," "second," and the like are used merely to distinguish one description from another, and are not to be construed as indicating or implying relative importance.
The following detailed description of embodiments of the invention refers to the accompanying drawings.
In the microfluid technology in the prior art, the centrifugal microfluid technology can effectively overcome the integration technical problem brought by external drive, micropump and the like. Centrifugal microfluidics thus provides the motive force for microfluidics through rotation of a disk-shaped microfluidic chip. The centrifugal microfluid technology transports cells through sedimentation force, and has the advantages of reliable cell transportation mode, high liquid control stability, no dependence on fluid properties (viscosity, pH, electric conductivity and the like) and the like. Centrifugal microfluidic technology is therefore a reliable way of performing cell manipulation and analysis functions.
The wireless power supply mode is realized by two non-contact electromagnetic coils which are mutually coupled. When the cell imaging acquisition module works, an alternating current signal is introduced into the transmitting coil and enters the receiving coil through electromagnetic coupling to generate alternating voltage, so that the electroosmosis electrode and the cell imaging acquisition module integrated on the chip are powered.
Currently, microfluidic technologies for cell capture and related cell manipulation have attracted attention in the related art. However, the centrifugal microfluidic technology for cell manipulation has not been developed effectively, and the existing centrifugal microfluidic chip mainly aims at whole blood biochemical analysis, immunological detection and gene amplification; the integration of the cell morphology analysis function is difficult to realize, and the functions of cell capture, high-resolution cell imaging and the like cannot be integrated.
Based on this, the present invention provides a centrifugal microfluidic chip integrated with cell microscopic function, as shown in fig. 1-8, wherein fig. 1 is a schematic structural diagram of a centrifugal microfluidic chip provided in an embodiment of the present invention; fig. 2 is an exploded view of the centrifugal microfluidic chip shown in fig. 1. Referring to fig. 1, the centrifugal microfluidic chip includes: the micro-fluidic chip comprises a micro-fluidic substrate 1, a bearing substrate 2, a wireless power supply receiving and image acquisition device 3 and a wireless power supply transmitting device 4 which are oppositely arranged.
It should be noted that the carrier substrate 2 has two functions: one is a groove structure on the sealed (or packaged) microfluidic substrate 1 to form a closed functional system. The second is a bearing concentric ring super lens array, a cell sorting electrode pair 16, a cell suspension ring electrode array 15, an electrode lead and other related structures. In this embodiment, the specific materials of the microfluidic substrate 1 and the carrier substrate 2 are not limited. Alternatively, the microfluidic substrate 1 and the carrier substrate 2 are hard PMMA (polymethyl methacrylate) plates, which are commonly used materials for processing microfluidic chips (such as PC and PMMA). The processing method of the microfluidic structure on the microfluidic chip made of the polymer material comprises hot pressing, laser processing and the like. And the hard polymer PMMA has better light transmission and good compatibility with the micro-fluidic structure processing technology. The material of the superlens array 14 may be titanium dioxide, which has a high refractive index and has a strong modulation effect on the propagation of light waves. The embodiment of the present invention does not limit the specific form of the image capturing unit 21, as long as the cell image information can be captured, processed and stored. Referring to fig. 2, optionally, in the embodiment of the present invention, the image capturing unit 21 is a CMOS image sensor, and the CMOS image sensor is connected to the control circuit module 22 through a lead 20. The surface of the CMOS image sensor is coated with a filter layer, and the filter layer is used for filtering stray light entering the CMOS image sensor. The filter layer enables the super lens to work at the resonance wavelength of the super lens, and stray light is filtered out, so that images acquired by the image sensor are clearer. The CMOS image sensor is a typical image sensor, and has a large resolution and a large model selection space, and is more convenient to mount on the carrier substrate 2.
Referring to fig. 2, the surface of the microfluidic substrate 1 facing the carrier substrate 2 is provided with a groove structure, the microfluidic substrate 1 is hermetically connected to the carrier substrate 2, the groove structure forms a plurality of cavities, the plurality of cavities at least include a plurality of cell suspension cavities 6, and the cell suspension cavities 6 correspond to the superlens arrays 14 one to one. The microfluidic substrate 1 comprises a through hole structure for mounting, venting and letting the microfluid into the groove structure. The plurality of cavities and the through hole structure form two parallel functional units. The microfluidics may be such that the surface of the sample liquid carrier substrate 2 facing the microfluidic substrate 1 is provided with a plurality of superlens arrays 14 and a plurality of cell suspension ring electrode arrays 15. The cell suspension annular electrode arrays 15 and the super lens arrays 14 are arranged in a one-to-one correspondence mode, one cell suspension annular electrode array 15 is arranged on the outer side of one super lens array 14 in a surrounding mode, and the cell suspension annular electrode array 15 is arranged in the cell suspension cavity 6. The surface of the carrier substrate 2 facing away from the microfluidic substrate 1 is provided with a plurality of image acquisition unit mounting cavities 18 and control circuit module mounting cavities 19.
The wireless power receiving and image capturing device 3 includes a plurality of image capturing units 21, a control circuit module 22, and a wireless power receiving module 23. The image acquisition unit 21 is connected with the control circuit module 22, and the control circuit module 22 is connected with the wireless power supply receiving module 23. The image acquisition unit 21 is embedded in the image acquisition unit installation cavity 18, and the control circuit module 22 is embedded in the control circuit module installation cavity 19.
Referring to fig. 2 and 4, in an embodiment, the plurality of cavities further includes two cell sedimentation cavities 8, two sets of cell sorting electrode pairs 16 are further disposed on the surface of the supporting substrate 2 facing the microfluidic substrate 1, the two sets of cell sorting electrode pairs 16 are disposed in one-to-one correspondence with the two cell sedimentation cavities 8, and the cell sorting electrode pairs 16 are disposed in the two cell sedimentation cavities 8. The sorting electrode pair 16 is applied with voltage while the chip rotates, so that the change of a local flow field is caused, and the sensitivity of the sorted biological particles to the flow field due to the characteristics of size and the like drives the cells with different sizes and forms different motion tracks to enter the set cell suspension cavity 6. Therefore, the difference of movement tracks among cells is increased by arranging the sorting electrode pair 16, so that the accuracy of different cells entering different set sorting cavities in the cell sedimentation cavity 8 is improved.
Referring to fig. 2, in the present embodiment, in order to implement operations such as cell sorting, the plurality of cavities further includes a sample injection cavity 5, a cell precipitation cavity 8, and a waste liquid cavity 7. The through hole structure comprises a sample inlet hole 9, a sample inlet cavity vent hole 10, a waste liquid cavity vent hole 13 and a mounting hole 11. The sample inlet hole 9 and the sample inlet cavity vent hole 10 are respectively positioned at two sides of the top end of the sample inlet cavity 5 (arranged in a central symmetry way in the figure). The sample inlet hole 9 and the sample inlet cavity vent hole 10 are both communicated with the sample inlet cavity 5. The waste liquid chamber vent 13 communicates with the waste liquid chamber 7. The sample feeding cavity 5 and the cell sedimentation cavity 8 are connected through a micro valve 12. The cell suspension annular electrode array 15 is arranged in the cell suspension cavity 6. The cell precipitation chamber 8 is connected to the waste liquid chamber 7 through a waste liquid chamber microchannel 24.
It should be noted that, because of the microfluidic function requirements: the functions related to cell manipulation in the microchannel are performed in the cell precipitation chamber 8 and the microchannel, and a small depth is required. And the waste liquid cavity 7 is filled with liquid, and the volume of the liquid is relatively large, so that the depth is increased, and the integration level and the compactness of the structure are improved. Optionally, in this embodiment, the waste liquid chambers 7 have the same first depth, and the sample injection chamber 5, the cell precipitation chamber 8 and the waste liquid chamber micro-channel 24 have the same second depth, wherein the first depth is greater than the second depth.
In this embodiment, the control circuit module 22 adopts a wireless power supply mode.
It should be noted that the superlens array 14 on the carrier substrate 2 is located at the position of the cell suspension chamber 6 of the microfluidic substrate 1. In the centrifugal microfluidic chip, the position of the superlens array 14 corresponds to a cell capturing microstructure in the cell suspension cavity 6, and after the cells are captured by the microstructure, the cell morphology is recorded through high-resolution imaging based on the superlens array 14, so that the image acquisition unit 21 acquires relevant cell morphology and mechanical information.
The following describes the structure of the centrifugal microfluidic chip provided in this embodiment specifically; referring to fig. 1 to 8, a centrifugal microfluidic chip is shown in fig. 1, and includes a hard polymer PMMA plate (i.e., a microfluidic substrate) 1, a hard polymer PMMA substrate (i.e., a carrier substrate) 2, a wireless power receiving and image acquiring device 3, and a wireless power transmitting device 4. As shown in fig. 2, a rigid polymer PMMA sheet 1 is attached to the surface of a rigid polymer PMMA substrate 2 to form a closed cavity. As shown in fig. 4, there are 8 sets of superlens arrays 14 on the rigid polymer PMMA substrate 2, which are positioned corresponding to the cell suspension chamber 6 of the rigid polymer PMMA substrate 1. The CMOS image sensor 21 and the control circuit module 22 in the wireless power supply receiving and image acquisition device 3 are embedded in the corresponding mounting cavities on the hard polymer PMMA substrate 2. The hard polymer PMMA substrate 2 has the diameter of 80mm, the thickness of 1mm and the diameter of 4mm of a mounting hole 11. The superlens array 14 is a concentric ring topology. The superlens array 14 is made of a titanium dioxide material. The superlens array 14 has a high numerical aperture, and specifically, the numerical aperture of the superlens array 14 is 0.9, so that light focusing close to a diffraction limit is realized, and high-resolution imaging is further realized. The superlens array 14 is shown in fig. 7 in a plan view and in fig. 8 in a sectional view, and the outer ring of the superlens array 101 has a diameter of 36 μm and a thickness of 600 nm. The depths of the image acquisition unit installation cavities 18 and the control circuit module installation cavity 19 on the other side of the hard polymer PMMA substrate 2 are both 1 mm.
The rigid polymer PMMA substrate 1 had a diameter of 80mm and a thickness of 2 mm. The depth of the sample injection cavity 5 and the waste liquid cavity 7 at one side of the hard polymer PMMA sheet 1 is 500 μm. The depth of the waste liquid chamber microchannel 24 and the cell suspension chamber 6 is 50 μm, and the width of the microvalve 12 between the sample introduction chamber 5 and the cell sedimentation chamber 8 is 50 μm. The cell suspension chamber 6 had 8 cells in total. The diameter of a sample inlet hole 9 on the hard polymer PMMA substrate 1 is 2 mm. The diameter of the air vent 10 of the sample injection cavity is 2mm, and the diameter of the air vent 13 of the waste liquid cavity is 2 mm.
The image capturing device 3 is formed by connecting a CMOS image sensor 18 and a control circuit module 19 via module circuit leads (not shown). The centrifugal micro-fluidic chip is powered by the wireless power supply transmitting device 4 through the wireless power supply receiving module 23.
The centrifugal microfluidic chip provided by the invention comprises a bearing substrate 2, a microfluidic substrate 1, a wireless power supply receiving and image acquisition device 3 and a wireless power supply transmitting device 4. The carrier substrate 2 includes a plurality of superlens arrays thereon. The microfluidic substrate 1 comprises a plurality of cell suspension cavities 6, and the cell suspension cavities 6 and the superlens arrays 14 are arranged in a one-to-one correspondence manner. The cell is sorted by the cell sedimentation cavity 8 and the cell sorting electrode pair 16 on the microfluidic substrate 1, and the cell is captured; the cell is suspended and focused by the cell suspension annular electrode array 15, and then imaged by the super lens array and the image acquisition unit 21, so that the centrifugal microfluidic chip has the functions of cell sorting, high-resolution cell imaging and the like.
In the centrifugal micro-fluidic chip, the cell suspension annular electrode array 15 is arranged outside the cell suspension cavity 6, after the centrifugal micro-fluidic chip stops rotating, a flow field which circularly flows can be formed in the cell suspension cavity 6 by applying voltage to the cell suspension annular electrode array 15, and cells are localized at the center of the annular flow field, namely in the optimal imaging area of the super lens, so that the imaging effect is enhanced.
The embodiment of the invention also provides an application method of the centrifugal microfluidic chip, which explains the use of the centrifugal microfluidic chip, so that the centrifugal microfluidic chip can be used for sorting cells and imaging the cells at high resolution. The application method of the centrifugal microfluidic chip comprises the following steps:
s11, providing a centrifugal microfluidic chip, a sample solution and a buffer solution, wherein the sample solution comprises a plurality of cells, and the buffer solution is a PBS buffer solution.
And S12, adding the buffer solution into the centrifugal micro-fluidic chip, and rotating the centrifugal micro-fluidic chip according to a first preset rotating speed and a first preset time.
And S13, adding the sample liquid into the centrifugal microfluidic chip, and rotating the centrifugal microfluidic chip according to a second preset rotating speed and a second preset time.
S14, when the cells in the sample liquid enter the cell sedimentation cavity, stopping rotating the centrifugal microfluidic chip, applying a preset voltage to the cell suspension annular electrode array on the bearing substrate, and after the cells in the sample liquid are captured by the cell suspension cavity, acquiring the image information of the captured cells through the image acquisition unit.
In one embodiment, the application method of the centrifugal microfluidic chip further comprises the following steps: and applying preset voltage to the cell sorting electrode pair on the bearing substrate while rotating the centrifugal micro-fluidic chip according to a second preset rotating speed and a second preset time.
The specific process is as follows:
the centrifugal micro-fluidic chip applies buffer solution to the sample injection cavity 5 from the sample injection hole 9. Specifically, the buffer is PBS buffer. The centrifugal microchip was mounted on a centrifugal motor through the mounting hole 11, and the chip was rotated at 3000 rpm for 1 minute. Buffer enters the cell precipitation chamber 8 through the micro valve 12 and fills it, and excess buffer enters the waste chamber 7 through the waste chamber microchannel 24. Blood is applied into the sample injection cavity 5 from the sample injection hole 9, and the applied blood will flow to the outlet of the sample injection cavity 5 and then stop due to size expansion. The chip is rotated at the rotating speed of 3000 rpm for 2 minutes, the blood in the sample injection cavity 5 enters the cell sedimentation cavity 8, the blood cells are classified and respectively enter different cell suspension cavities 6, then are captured by the cell capturing micro-structure array, and the redundant liquid enters the waste liquid cavity 7 through the waste liquid cavity micro-channel 24. The exciting light passes through the cell suspension cavity 6 from the bottom surface of the chip and enters the CMOS image sensor, and the cell image information obtained by the CMOS image sensor is recorded by the control circuit module 22.
Referring to fig. 9, the present invention further provides a method for manufacturing a centrifugal microfluidic chip; the method comprises the following steps:
step S101: the method comprises the steps of providing a bearing substrate body, a micro-fluidic substrate body, a wireless power supply receiving and image acquisition device and a wireless power supply transmitting device.
Step S102: the bearing substrate body comprises a first surface and a second surface which are oppositely arranged, a plurality of image acquisition unit installation cavities, a wireless power supply receiving module installation cavity and a control circuit module installation cavity are manufactured on the first surface of the bearing substrate body, and a cell suspension annular electrode array lead is manufactured at a preset position. Two sets of cell sorting electrode pairs are manufactured on the second surface of the bearing substrate body, and a cell suspension annular electrode array pair and a super lens array with a concentric ring topological structure are manufactured at preset positions to obtain the bearing substrate.
Specifically, two sets of cell sorting electrode pairs and cell suspension annular electrode arrays are processed on the surface of a bearing substrate body, wherein the surface is provided with a plurality of super lens arrays, and the super lens arrays, the cell sorting electrode pairs and the cell suspension annular electrode arrays are covered and protected through an evaporation process of a Parylene material.
Step S103: and manufacturing two sets of groove structures in one surface of the microfluidic substrate body, and manufacturing a through hole structure in the microfluidic substrate body to obtain the microfluidic substrate.
Step S104: the wireless power supply receiving and image collecting device comprises a plurality of image collecting units, a control circuit module and a wireless power supply receiving module, wherein the image collecting units are connected with the control circuit module, and the control circuit module is connected with the wireless power supply receiving module. The image acquisition unit is installed in the image acquisition unit installation cavity, the wireless power supply receiving module is installed in the wireless power supply receiving module installation cavity, and the wireless power supply transmitting device and the wireless power supply receiving module are correspondingly arranged.
Step S105: and assembling and sealing the surface of the bearing substrate with the super lens array and the surface of the microfluidic substrate with the groove structure to obtain the centrifugal microfluidic chip.
In one embodiment, the method for manufacturing the centrifugal microfluidic chip further includes the following steps:
manufacturing a cell sorting electrode pair lead at a preset position on the first surface of the bearing substrate body; two sets of cell sorting electrode pairs are manufactured on the second surface of the bearing substrate body.
The steps of fabricating the superlens array on the carrier substrate are shown in fig. 10A-10D, and include:
step one, gluing, namely cleaning the bearing substrate 2, and spin-coating electron beam photoresist 1011 with the thickness of 650 nm-700 nm on the bearing substrate;
step two, hardening, namely placing the bearing substrate 2 coated with the electron beam photoresist 1011 on a hot plate at 180 ℃ for drying the photoresist for 5 minutes;
step three, photoetching, namely performing electron beam direct writing exposure according to a concentric ring section of the superlens shown in the figure 6 by adopting an electron beam photoetching process;
step four, development, using MIBK (methyl isobutyl ketone): developing with IPA (isopropyl alcohol) developer solution in the ratio of 1:3 (the volume ratio), cleaning in ethanol for 30 seconds after the development is finished, and drying by using compressed air;
depositing, namely depositing a titanium dioxide layer 1012 with the thickness of 600 nanometers on the bearing substrate 2 with the electron beam photoresist structure by adopting an atomic layer deposition process;
and step six, stripping, namely placing the bearing substrate 2 deposited with the titanium dioxide layer 1012 in an acetone solution, uniformly shaking for 30 seconds to strip the electron beam photoresist 1011, taking out the bearing substrate 2, placing the bearing substrate in ethanol for cleaning for 30 seconds, drying by compressed air, and finally forming the superlens array on the bearing substrate 2.
The production of the hard polymer PMMA sheet 2 comprises the following steps:
polishing, namely polishing the surfaces of two brass substrates by adopting a mechanical polishing process;
and secondly, milling, namely processing hot-pressing molds corresponding to structures on two sides of the polymer PMMA sheet on two brass substrates (comprising a first brass substrate and a second brass substrate) respectively by adopting a high-precision numerical control milling process, and polishing, wherein the height of a protruding structure corresponding to a sample injection cavity 5 on the first brass substrate and the height of a protruding structure corresponding to a waste liquid cavity 7 on the first brass substrate are 500 micrometers, and the height of protruding structures corresponding to the rest of microchannels and cavities is 50 micrometers. The width of the convex structure corresponding to the micro valve 12 between the sample feeding cavity 5 and the cell sedimentation cavity 8 is 50 μm. The total number of the protruding structures corresponding to the cell suspension cavity 6 is 8, and the heights of the protruding structures corresponding to the image acquisition unit installation cavity 18 and the control circuit module installation cavity 19 on the second brass substrate are all 1 mm.
Step three, hot pressing, namely forming a hot pressing die by using a first brass substrate and a second brass substrate, and hot pressing a hard polymer PMMA circular plate with the thickness of 2.5mm to obtain a hard polymer PMMA plate 2, wherein the hot pressing temperature is 135 ℃, the pressure maintaining time is 30 minutes, and the pressure is 50 kilopascals;
and fourthly, drilling, namely machining a sample inlet with the diameter of 2mm, a sample injection cavity vent hole with the diameter of 2mm and a waste liquid cavity vent hole with the diameter of 2mm on the hard polymer PMMA plate by adopting a numerical control machine.
Bonding and assembling:
step one, spin coating, namely coating a filter layer on the surface of the CMOS image sensor.
And step two, a lead wire is used for connecting the corresponding pin between the CMOS image sensor and the control circuit module to form the wireless power supply receiving and image acquisition device 3.
Bonding, namely spin-coating alcohol and acetone mixed solution on the hard polymer PMMA plate, then bonding the hard polymer PMMA plate with the bearing substrate 2, and aligning the superlens array on the bearing substrate 2 with the cell suspension cavity 6 on the hard polymer PMMA plate during bonding;
and step four, assembling, namely respectively embedding the CMOS image sensor and the control circuit module into corresponding installation cavities on the hard polymer PMMA plate, and dispensing and packaging.
The cavity on the microfluidic substrate is manufactured by a micron-scale process, and the superlens array on the bearing substrate is manufactured by a nanometer-scale process.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (7)
1. A centrifugal microfluidic chip for cell deposition and cell capture, comprising:
the device comprises a microfluidic substrate, a bearing substrate, a wireless power supply receiving and image collecting device and a wireless power supply transmitting device which are oppositely arranged; the wireless power supply receiving and transmitting device is two mutually coupled non-contact electromagnetic coils;
the surface of the microfluidic substrate facing the bearing substrate is provided with a groove structure, the microfluidic substrate is hermetically connected with the bearing substrate, the groove structure forms a plurality of cavities, the plurality of cavities at least comprise a plurality of cell suspension cavities, and the cell suspension cavities correspond to the superlens arrays one by one; the microfluidic substrate comprises a through hole structure which is used for installing, ventilating and leading the microfluid to enter the groove structure; the plurality of cavities and the through hole structure form two parallel functional units;
the surface of the bearing substrate facing the microfluidic substrate is provided with a plurality of super lens arrays and a plurality of cell suspension annular electrode arrays; the cell suspension annular electrode arrays are arranged in one-to-one correspondence with the super lens arrays, one cell suspension annular electrode array is arranged on the outer side of one super lens array in a surrounding manner, and the cell suspension annular electrode arrays are arranged in the cell suspension cavity; the surface of the bearing substrate, which is far away from the microfluidic substrate, is provided with a plurality of image acquisition unit installation cavities and control circuit module installation cavities;
the wireless power supply receiving and image collecting device comprises a plurality of image collecting units, a control circuit module and a wireless power supply receiving module, wherein the image collecting units are connected with the control circuit module, and the control circuit module is connected with the wireless power supply receiving module; the image acquisition unit is embedded in the image acquisition unit mounting cavity, and the control circuit module is embedded in the control circuit module mounting cavity;
the multiple cavities at least comprise two cell sedimentation cavities, two sets of cell sorting electrode pairs which are centrosymmetrically arranged are further arranged on the surface of the bearing substrate facing the microfluidic substrate, the two sets of cell sorting electrode pairs are arranged in one-to-one correspondence with the two cell sedimentation cavities, and the cell sorting electrode pairs are arranged in the two cell sedimentation cavities.
2. The centrifugal microfluidic chip of claim 1, wherein the plurality of chambers further comprises a sample injection chamber, a cell precipitation chamber, and a waste solution chamber;
the through hole structure comprises a sample inlet hole, a sample inlet cavity vent hole, a waste liquid cavity vent hole and a mounting hole;
the sample inlet hole and the sample injection cavity vent hole are respectively positioned at two sides of the top end of the sample injection cavity, and the sample inlet hole and the sample injection cavity vent hole are both communicated with the sample injection cavity;
the waste liquid cavity vent hole is communicated with the waste liquid cavity;
the sample injection cavity is connected with the cell sedimentation cavity through a micro valve;
the cell precipitation cavity is connected with the waste liquid cavity through a waste liquid cavity micro-channel.
3. The microfluidic centrifugal chip of claim 2, wherein the sample inlet chamber and the waste chamber have a first depth, and the cell sedimentation chamber and the waste chamber microchannel have a same second depth, wherein the first depth is greater than the second depth.
4. The microfluidic chip according to claim 1, wherein the image capturing unit is a CMOS image sensor, a surface of the CMOS image sensor is coated with a filter layer, and the filter layer is used for filtering out stray light entering the CMOS image sensor.
5. The microfluidic centrifugal chip of any one of claims 1-4, wherein the superlenses of the superlens array are in a concentric ring topology, and the numerical aperture of the superlenses is 0.9.
6. An application method of a centrifugal microfluidic chip, wherein the centrifugal microfluidic chip is as claimed in any one of claims 1 to 5, the application method comprising:
providing the centrifugal microfluidic chip, a sample solution and a buffer solution, wherein the sample solution comprises a plurality of cells, and the buffer solution is a PBS buffer solution;
adding a buffer solution into a centrifugal micro-fluidic chip, and rotating the centrifugal micro-fluidic chip according to a first preset rotating speed and a first preset time;
adding the sample liquid into the centrifugal microfluidic chip, and rotating the centrifugal microfluidic chip according to a second preset rotating speed and a second preset time;
when the cells in the sample liquid enter the cell sedimentation cavity, stopping rotating the centrifugal microfluidic chip, applying a preset voltage to the cell suspension annular electrode array on the bearing substrate, and after the cells in the sample liquid are captured by the cell suspension cavity, acquiring image information of the captured cells through the image acquisition unit;
also comprises the following steps: and applying a preset voltage to the cell sorting electrode pair on the bearing substrate while rotating the centrifugal micro-fluidic chip according to a preset rotating speed and preset time.
7. A method for manufacturing a centrifugal microfluidic chip, the method being used for manufacturing the centrifugal microfluidic chip according to any one of claims 1 to 5, the method comprising:
providing a bearing substrate body, a micro-fluidic substrate body, a wireless power supply receiving and image collecting device and a wireless power supply transmitting device;
the bearing substrate body comprises a first surface and a second surface which are oppositely arranged, a plurality of image acquisition unit installation cavities, a wireless power supply receiving module installation cavity and a control circuit module installation cavity are manufactured on the first surface of the bearing substrate body, and a cell suspension annular electrode array lead is manufactured at a preset position; manufacturing two sets of cell sorting electrode pairs on the second surface of the bearing substrate body, and manufacturing a cell suspension annular electrode array and a super lens array with a concentric ring topological structure at a preset position to obtain a bearing substrate;
manufacturing two sets of groove structures in one surface of the microfluidic substrate body, and manufacturing a through hole structure in the microfluidic substrate body to obtain a microfluidic substrate;
the wireless power supply receiving and image collecting device comprises a plurality of image collecting units, a control circuit module and a wireless power supply receiving module, wherein the image collecting units are connected with the control circuit module, the control circuit module is connected with the wireless power supply receiving module, the image collecting units are installed in an image collecting unit installation cavity, the wireless power supply receiving module is installed in a wireless power supply receiving module installation cavity, and the wireless power supply transmitting device and the wireless power supply receiving module are correspondingly arranged;
assembling and sealing the surface of the bearing substrate with the super lens array and the surface of the microfluidic substrate with the groove structure to obtain the centrifugal microfluidic chip;
also comprises the following steps:
manufacturing a cell sorting electrode pair lead at a preset position on the first surface of the bearing substrate body;
and manufacturing two sets of cell sorting electrode pairs on the second surface of the bearing substrate body.
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| CN105717186B (en) * | 2016-02-01 | 2018-06-29 | 常熟理工学院 | Swinging dielectrophoresis microfluidic analysis device |
| CN107398307B (en) * | 2016-05-18 | 2023-07-25 | 博奥生物集团有限公司 | Integrated micro-fluidic chip |
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