CN109913352B - Microfluidic device and method for controlling and capturing microparticles and cells based on non-contact dielectrophoresis force - Google Patents
Microfluidic device and method for controlling and capturing microparticles and cells based on non-contact dielectrophoresis force Download PDFInfo
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Abstract
The invention provides a microfluidic device for controlling and capturing microparticles and cells based on non-contact dielectrophoresis force, which comprises: a sample injection pump, a micro-injector, a micro-fluidic chip and a waste liquid collector which are connected in sequence; the micro-fluidic chip is formed by laminating a glass substrate layer and a polydimethylsiloxane chip layer, wherein the polydimethylsiloxane chip layer comprises: a sample injection port and a sample injection channel; a sheath flow sample inlet and a sheath flow sample inlet channel; the first liquid electrode channel and the second liquid electrode channel are respectively and independently formed into a closed ring shape and are arranged at two sides of the sample separation channel so as to apply a high-frequency high-voltage non-uniform electric field to the first liquid electrode channel and the second liquid electrode channel; a target product collection channel and a waste liquid collection channel; and a sample outlet. The invention also provides a method for capturing microparticles and cells based on non-contact dielectrophoresis force control. The device and the method provided by the invention have the advantages of high separation efficiency, simple operation, low cost and high flux.
Description
Technical Field
The invention relates to the field of biological detection, in particular to a microfluidic device and a method for controlling and capturing microparticles and cells based on non-contact dielectrophoresis force.
Background
Cancer is still one of the diseases with extremely high global mortality. In 2008, the cancer-dying population reaches 760 million (13% of the total number of deaths) and may exceed 1300 million by 2030. Early diagnosis and treatment of primary cancer and effective treatment of metastatic cancer are key to fighting cancer.
Circulating Tumor Cells (CTCs) are a type of Tumor cell that originates from a primary Tumor and enters the blood circulation system. The cells can metastasize through the blood circulation system and reach other organs to achieve tumor metastasis, so that the CTCs are closely related to the occurrence, development and metastasis of tumors. In view of the important role played by CTCs in tumor metastasis, we can monitor the effect and recurrence of tumor therapy in real time by detecting changes in CTCs.
Normal human 1mL of peripheral blood contains 5X 10 of the blood10Red blood cells, 7X 106White blood cell and 2.95X 106Platelets. Analyzing tumor cells from blood having such a complex composition is a great challenge. If the separation and enrichment of the CTCs can be realized, the number and the molecular characteristics of the CTCs can be analyzed, and a basis is provided for tumor diagnosis and typing and target drug treatment.
Enrichment of CTCs is achieved primarily based on the specific physicochemical properties of the target cells. Conventional enrichment methods include density gradient centrifugation, membrane filtration, microfluidic chip methods, and antibody-dependent separation methods. Among them, the FDA approved commercial CellSearchTM system (Veridex LLC) belongs to an antibody-dependent method for isolating circulating tumor cells. The system is used for enriching CTCs by specifically binding antibody-modified magnetic beads with Epithelial cell surface expression markers, namely Epithelial cell adhesion molecules (EpCAM). However, this detection method is currently only applied to breast, colon and Prostate cancers [ Miller MC, Doyle GV, Terstappen LW Significance of circulating tumor Cells Detected by the cell search System in Patients with Metastatic Breast color and State cancer. J Oncol,2010,617421], but the detection rate in lung cancer is low. Tanaka et al detected 125 lung Cancer patients by the CellSearch system, and CTCs (30.6%) were detected only in the peripheral blood of 38 patients [ Tanaka F, Yoneda K, et al. circulating tumor cells as a diagnostic marker in primary Cancer. J.Clin Cancer Res,2009,15(22):6980 ]. The capture efficiency of the immunomagnetic bead detection method mainly depends on the CTCs marker EpCAM, however, the expression of EpCAM antigen in different types of tumors is heterogeneous, and even in some tumors of non-epidermal origin, the expression is absent. Therefore, the detection method has the defects of low detection sensitivity, narrow application range, high cost, long consumed time and the like.
To solve these disadvantages, a method for separating target cells by using the difference in physical properties between circulating tumor cells and normal blood cells has been developed. In recent years, with the continuous improvement and development of microfluidic and micromachining technologies, it has become possible to realize microfluidic chip channels and apertures of precise dimensions. Therefore, the microfluidic chip method based on the cell size can obtain higher capture efficiency, and in addition, the microfluidic chip has the advantages of small volume, high analysis speed, low cost and the like. These methods are all beneficial to applying the microfluidic chip method to clinical application so as to make up for the defects of the existing clinical detection method. Many methods for detecting circulating tumor cells based on a microfluidic chip method are currently available, and patent document 1(CN 103642756a) discloses a method for capturing circulating tumor cells by modifying single-chain nucleotides on the surface of a microfluidic chip microchannel and combining the single-chain nucleotides with an antibody labeled with complementary nucleotides.
In the prior art, the capture scheme of the circulating tumor cells can be roughly classified into two categories, namely passive sorting and active sorting, wherein the passive sorting technology comprises methods such as microstructural filtration, field flow and hydraulic sorting, deterministic lateral migration, inertial sorting, bionic sorting, affinity sorting and the like; the active sorting technology comprises methods such as dielectrophoresis sorting, magnetic sorting, acoustic sorting, optical sorting and the like, wherein a dielectrophoresis sorting scheme is selected, the dielectrophoresis is different from the common electrophoresis, the application range of the dielectrophoresis is wide, and neutral particles without electricity are acted, so that the dielectrophoresis can be used for controlling and sorting neutral microparticles and cells. The dielectrophoresis force generated by dielectrophoresis is a non-contact force, so that the micro-particles and cells cannot be mechanically damaged in the process of manipulating the micro-particles and cells, and the dielectrophoresis force depends on the electrical parameters of the micro-particles and the cells to enable the micro-particles and the cells to perform polarization motion in a high-frequency high-voltage electric field, so that additional marking operation is not needed. The advantages of this scheme include: 1. neutral particles are acted on; 2. a non-contact force; 3. the method does not need to be marked, so that the dielectrophoresis scheme is adopted by the people to realize the control and capture of the microparticles and the cells.
However, in the conventional metal contact type dielectrophoresis chip, the electrode is in contact with the sample, the sample is polluted, electric heat on the electrode has a large influence on the sample, and the like, and meanwhile, the metal electrode must be deposited on the chip substrate firstly, and then aligned bonding is carried out, so that the process is complex, and the chip cost is high.
Disclosure of Invention
The invention aims to provide a microfluidic device and a method for controlling and capturing microparticles and cells based on non-contact dielectrophoresis force, so that the problems that a traditional metal contact dielectrophoresis chip pollutes a sample, the process is complex and the chip cost is high are solved.
In order to solve the technical problems, the invention adopts the following technical scheme:
according to a first aspect of the present invention, there is provided a microfluidic device for trapping microparticles and cells based on non-contact dielectrophoretic force manipulation, comprising: a sample injection pump, a micro-injector, a micro-fluidic chip and a waste liquid collector which are connected in sequence; the micro-fluidic chip is formed by laminating a glass substrate layer and a polydimethylsiloxane chip layer, wherein the polydimethylsiloxane chip layer comprises: the sample injection port is connected with a sample injection channel; the sheath flow sample inlet is led out from the sheath flow sample inlet and is divided into two sheath flow sample inlet channels, and the tail ends of the two sheath flow sample inlet channels are respectively converged with the sample inlet channels at two sides of the sample inlet channel to form a sample separation channel; first and second liquid-electrode channels each independently in a closed loop shape disposed on both sides of the sample separation channel, the first liquid-electrode channel comprising: first electrode liquid introduction port and the most advanced wave structure of channel, the second liquid electrode channel includes: the second electrode liquid injection port and the channel tip gentle structure are arranged on the two opposite sides of the sample separation channel respectively, so that a high-frequency high-voltage non-uniform electric field is applied to the sample separation channel; the sample separation channel is led out from the tail end of the sample separation channel and is divided into a target product collection channel and a waste liquid collection channel which are formed in two parts respectively; and the sample outlet is respectively connected with the target product collecting channel and the waste liquid collecting channel.
According to the microfluidic chip in the microfluidic device provided by the invention, the first liquid electrode channel and the second liquid electrode channel are respectively arranged at two opposite sides of the sample separation channel, on one hand, the tip of the electrode is away from the edge of the sample separation channel by a certain distance and is separated by the solidified PDMS, so that the microfluidic chip is called a non-contact liquid channel electrode; on the other hand, the main structures of the first and second liquid electrode channels are basically consistent, but the shape of one side of the channel tip part is a wave-shaped bulge, and the shape of one side of the channel tip part is a gentle extension, so the channel is also called a local asymmetric electrode. The working principle is as follows: according to the tip effect, the smaller the curvature radius of the surface of the electrode is, the larger the surface charge density is, the denser the power line is, and the higher the electric field intensity is; and on the contrary, the lower the electric field intensity is, so that a high-frequency high-voltage non-uniform electric field is applied to the sample separation channel, the sample polarization in the sample separation channel is induced, and then the deflected sample is sorted.
Compared with the dielectrophoresis chip disclosed in the prior art, the dielectrophoresis chip adopts the contact type metal electrode, the non-contact type local asymmetric liquid electrode channel is creatively adopted to replace the traditional metal electrode, the manufacturing process of the chip is greatly simplified while the effect is achieved, the manufacturing cost and the bonding precision requirement of the chip are reduced, and the pollution and the electric heating influence of the metal electrode on a sample are avoided.
The height of the whole microfluidic chip is uniform, preferably the height is 30-50 μm, and most preferably 40 μm.
Preferably, in the microfluidic chip, a cylindrical column for supporting the channel is arranged in the liquid storage channel with a larger electrode part, and the diameter of the cylindrical column is about 100 μm.
Preferably, the sample injection channel is arranged in the center of a serpentine channel, and the sheath flow sample injection channels are respectively arranged at two sides far away from the sample injection channel and then gradually close to merge with the sample injection channel to form a sheath flow structure.
Before merging, the widths of the sample feeding channel and the sheath flow feeding channel are gradually narrowed.
Preferably, the width of the sample injection channel is 150-200 μm, the final width becomes 30-40 μm when the width of the sheath flow structure part is gradually narrowed, the width of the sheath flow sample injection channel is 100-120 μm when the width of the sheath flow structure part is gradually narrowed, and the final width becomes 20-30 μm.
Most preferably, the sample introduction channel is 200 μm wide and gradually narrows to a final width of 40 μm in the sheath flow structure portion, and the sheath flow introduction channel is 100 μm wide and gradually narrows to a final width of 30 μm in the sheath flow structure portion.
The first liquid electrode channel further comprises: first electrode liquid storage district and first electrode liquid buffer, the second liquid electrode channel still includes: a second electrode solution storage area and a second electrode solution buffer area.
The first electrode liquid storage area and the second electrode liquid storage area play a buffering role, and the phenomenon that the subsequent shaking is caused when the metal electrodes are inserted into the first electrode liquid injection port and the second electrode liquid injection port, so that the electrode liquid in the electrode channel is cut off and the electric conduction of the liquid electrodes is influenced is prevented.
The channel tip wavy structure is a section of channel with a wavy shape, and the channel tip gentle structure is a section of channel with a straight line shape.
The microfluidic device further comprises a plurality of target product reservoir chambers connected to the target product collection channels. It will be appreciated that different numbers or sizes of target product reservoirs may be provided, for example three, four or six, depending on the actual requirements.
The sample injection pump and the micro-injector which are connected in sequence are used as a power system in the micro-fluidic device provided by the invention, and the sample injection flow rate can be accurately controlled by adopting a positive pressure sample injection mode.
And a step-by-step flow distribution structure is also arranged between the target product collecting channel and the target product liquid storage cavity.
The micro-fluidic device also comprises electrodes respectively inserted into the first electrode liquid sample inlet and the second electrode liquid sample inlet, a high-voltage amplifier in signal connection with the electrodes, a function generator and a computer.
According to a second aspect of the present invention, there is provided a method for capturing microparticles and cells based on non-contact dielectrophoretic force manipulation, comprising the steps of: 1) providing a microfluidic device for trapping microparticles and cells based on non-contact dielectrophoretic force manipulation as described above; 2) introducing washing liquor to wash the microfluidic chip, simultaneously introducing sheath flow liquid and a sample into the microfluidic chip, and after stable sheath flow liquid in the microfluidic chip is formed, opening an external circuit switch to control and capture microparticles or cells; and 3) observing the movement of the microparticles and the cells in the electric field in real time under a microscope, and analyzing the optimal physical parameters aiming at different microparticles or cells.
Specifically, step 1) further comprises: and sucking the sample into a micro-syringe, connecting the micro-syringe to a sample injection pump, and connecting the micro-syringe with the micro-fluidic chip through a thin tube.
Step 2) also includes: and placing the microfluidic chip in a vacuum pan for vacuumizing, and then sucking the DEP buffer solution by using a gun head and respectively injecting the DEP buffer solution into the first electrode solution injection port and the second electrode solution injection port.
Step 2) also includes: the chip is washed by the DEP buffer solution, namely, the DEP buffer solution is firstly subjected to a sample introduction process through the sample introduction port and the sheath solution introduction port, so that the DEP buffer solution is filled in all channels, a washing and infiltration effect is realized, and the subsequent sample introduction is facilitated.
And step 2) also comprises adjusting the flow rate of a sample injection pump to 10mL/h-30mL/h, so that the sample injection is started together with the buffer solution.
According to the difference of dielectric parameters and physical dimensions of the sample, the purpose of differentially deflecting and sorting the sample can be achieved by changing the condition of an applied electric field, optimizing the dielectric parameters of a buffer solution, adjusting the flow rate of a channel, the dimension of a main channel, the dimension of a liquid channel and the distance between the liquid channel and the main channel.
According to the present invention, microparticles refer to micron-sized electrically neutral particles, such as polystyrene microspheres and the like, and cells may include circulating tumor cells and the like.
When the method provided by the invention is used for capturing the polystyrene microspheres, the working principle is as follows: the electrically neutral polystyrene microsphere is subjected to dielectric polarization in a high-frequency non-uniform electric field, namely a small amount of free charges in the polystyrene microsphere are rearranged under the action of an external electric field to form a dipole moment, which can be represented by two charges with the same charge quantity but opposite polarities, when the dipole moment is positioned in the non-uniform electric field, a net force is generated due to the difference of local electric field intensities on two sides of the particle, the net force is called dielectrophoresis force, and the polystyrene microsphere can move in a translation mode under the net force through optimized conditions and enters a target product collecting channel to be collected.
When the method provided by the invention is used for capturing circulating tumor cells, the sample is washed with PBS and shaken and centrifuged twice before being injected, and is resuspended in DEP buffer (8.5% sucrose [ wt/vol ], 0.3% glucose [ wt/vol ]). Then the mixture was taken up in a 100. mu.L microinjector. The process of sampling needs to use two parallel channels of a sampling pump. After the sample introduction starts and the sheath flows out stably, the subsequent dielectrophoresis operation can be carried out.
The micro-fluidic chip for capturing the circulating tumor cells based on the non-contact dielectrophoresis can be combined with fluorescent staining identification to construct a method for detecting and sorting the circulating tumor cells, so that the circulating tumor cells can be dynamically detected and captured in the flowing process. The microfluidic device for capturing the circulating tumor cells based on the non-contact dielectrophoresis technology provided by the invention greatly improves the capturing capability of the circulating tumor cells, and the cost of the microfluidic chip is low, so that on one hand, a silicon wafer mould is made and then can be recycled; on the other hand, the electrode part in the chip is replaced by the channel, so that the step of depositing a metal electrode on the substrate before lifting is omitted, and meanwhile, the electrode channel and the sample channel are positioned in the same layer, so that the electrode channel can be etched out together when the sample channel is etched on the mold, the process is simple, and the alignment procedure of chip bonding in the later period is omitted.
In a word, the invention provides the microfluidic device and the method which have the advantages of high separation efficiency, simple operation, low cost and high flux and apply active force to control and capture the circulating tumor cells in the flowing process.
Drawings
Fig. 1 is a schematic view showing the overall structure of a microfluidic device provided according to a preferred embodiment of the present invention;
fig. 2 is a schematic diagram showing the structure of a microfluidic chip in the microfluidic device;
FIG. 3 is an enlarged detail schematic view of the sheath flow structure shown in FIG. 2;
FIG. 4 is a schematic view of the structure of FIG. 2 after the liquid electrode channel portion is not filled with the conductive liquid and filled with the conductive liquid;
FIG. 5 is a comparison graph of the deflection of microspheres of different sizes when different electric field parameters are applied during the injection of the microspheres in example 2;
FIG. 6 is a graph showing the results of the capture of circulating tumor cells H446 after the electrodes are activated when the sample injection is set to the circulating tumor cells H446 in example 3;
FIG. 7 is a simulation diagram of the potential electric field distribution of the dielectrophoresis region shown in FIG. 6.
Detailed Description
The present invention will be further described with reference to the following specific examples. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present invention.
According to a preferred embodiment of the present invention, there is provided a microfluidic device for capturing microparticles and cells based on non-contact dielectrophoretic force manipulation, as shown in fig. 1, including: the device comprises a sample pump 100, a micro-injector 200, a micro-fluidic chip 300, a waste liquid collector 400, a high-pressure amplifier 500, a function generator 600 and a computer 700 which are connected in sequence.
Specifically, the structure of the microfluidic chip 300 is shown in fig. 2, and the microfluidic chip 300 is formed by attaching a Polydimethylsiloxane (PDMS) chip layer 301 and a glass substrate layer 302.
Referring to fig. 2, 3, and 4, the polydimethylsiloxane chip layer 301 includes: the sample injection port 1 is connected with the sample injection channel 2; the sheath flow sample inlet 3 is led out from the sheath flow sample inlet 3 and is divided into two sheath flow sample inlet channels 4, and the tail ends of the two sheath flow sample inlet channels 4 are respectively converged with the sample inlet channel 2 at two sides of the sample inlet channel 2 to form a sample separation channel 5; the first and second liquid electrode channels which are independent in a closed ring shape and arranged on both sides of the sample separation channel 5, have the same general structure, and both comprise: the difference is that the first liquid electrode channel further comprises a channel tip wavy structure 9, and the second liquid electrode channel further comprises a channel tip gentle structure 10. Wherein, the channel tip wavy structure 9 and the channel tip gentle structure 10 are respectively arranged at two opposite sides of the sample separation channel 5 to apply a high-frequency high-voltage non-uniform electric field to the sample separation channel 5. The polydimethylsiloxane chip layer 301 also includes: a target product collecting channel 11 and a waste liquid collecting channel 12 which are led out from the tail end of the sample separation channel 5 and are divided into two parts; and outlet ports 13, 13' connected to the target product collecting channel 11 and the waste liquid collecting channel 12, respectively.
According to the preferred embodiment, a stepwise flow-dividing structure 14 and four target product storage cavities 15 are further designed between the target product collecting channel 11 and the sample outlet 13, as shown in fig. 2. It is understood that the target product reservoirs 15 may be designed in different numbers or different sizes, e.g. three, four or six, etc., according to the actual requirements.
According to the preferred embodiment, as shown in fig. 3, the width of the sample introduction channel 2 and the width of the sheath flow introduction channel 4 are gradually narrowed before merging. Preferably, the width of the main body part of the sample injection channel 2 is 150-200 μm, the final width becomes 30-40 μm when the main body part is gradually narrowed, and the width of the main body part of the sheath flow sample injection channel 4 is 100-120 μm when the main body part is gradually narrowed, and the final width becomes 20-30 μm when the main body part is gradually narrowed.
Preferably, the sample introduction channel 2, the electrode solution buffer 7, the target product collection channel 11 and the waste liquid collection channel 12 are all arranged in a serpentine channel, as shown in fig. 2.
Example 1 preparation of microfluidic chip
1.1, firstly, drawing a required structural graph by using AutoCAD drawing software aiming at a structural part of a microfluidic chip, manufacturing a mask, carrying out glue coating photoetching, reactive ion etching, photoresist removing and cleaning, glue coating photoetching and deep reactive ion etching by using a four-inch monocrystalline silicon wafer as a substrate, and etching the height of a chip channel to obtain a silicon wafer mold with a microstructure.
1.2 placing a silicon wafer mould and an open centrifugal tube filled with 10 mu L of fluorosilane in a vacuum drying box, vacuumizing to negative pressure to vaporize the fluorosilane, and standing the mould in fluorosilane steam for 5-6 hours. In the ventilation place, the drying oven was opened, and after ventilation for 1 hour, the silicon wafer was taken out. The purpose of this step is to deposit a layer of organic matter on the surface of the silicon chip, which is convenient for the subsequent manufacture of the PDMS chip.
1.3, according to the weight ratio of 10: 1, respectively weighing PDMS prepolymer and curing agent, then mixing and stirring uniformly, placing in a vacuum drying oven for vacuumizing, and standing for 30min under negative pressure. And after the PDMS is basically free of bubbles, pouring the PDMS on a silicon wafer mould, standing for 30min, and then putting the PDMS in a 65 ℃ oven to be heated for 1 h. And finally, stripping the cured PDMS chip layer from the mold, punching according to the sample inlet and outlet on the pattern, and cutting off the excessive part outside the structure. And finally, putting the structural surface of the PDMS chip layer 301 upwards and the glass substrate layer 302 into a plasma cleaning machine for cleaning for 1min, and quickly attaching the PDMS chip layer and the glass substrate layer together after being taken out, thereby completing the packaging of the microfluidic chip 300.
Example 2 the microfluidic chip prepared in example 1 was used to perform a deflective sorting of polystyrene microspheres of different sizes
The encapsulated micro-fluidic chip 300 of the embodiment 1 is placed in a vacuum pan and vacuumized for 30min, then the micro-fluidic chip 300 is taken out, a pipette is used for sucking 100 mu L of DEP conductive solution, then the pipette head is inserted into an electrode solution injection port 6 on the micro-fluidic chip 300, the micro-fluidic chip is kept still for 10min, and the DEP conductive solution is respectively sucked into a first liquid electrode channel and a second liquid electrode channel by depending on the negative pressure in the closed electrode channel. The effect after the liquid electrode channel portion is not filled with the conductive liquid and filled with the conductive liquid is shown at A, B in fig. 4.
It should be understood that the electrode material used herein may also be replaced by other well-conducting metallic materials, such as: copper, etc., and the platinum electrode is selected mainly because the platinum electrode is an inert electrode and is not easily oxidized.
The start button of the sample pump 100 is turned on, after two minutes, the liquid flow is stable, the sheath flow in the main channel is stable, the polystyrene microspheres stably flow into the waste liquid collecting channel 12, the switch of the function generator 600 is closed, the output parameters are adjusted to be 5V, the frequency is 100KHz, the ATA2161 switch is closed, the parameters are adjusted to be amplified by 200 times, and the output mode is differential output. It can be observed that after a short delay, the microspheres flowing through the sample separation channel 5 start to deflect towards the target product collection channel 11, the polystyrene microspheres flow into the end sample collection channels of the main channel and finally into the four sample collection chambers via the serpentine channels. The principle is that after an electrode switch is closed, liquid is used as an electrode, a high-frequency non-uniform electric field is applied to a main channel region clamped by the electrode, neutral microparticles flowing through the region are rapidly polarized, and then deflection is generated in the non-uniform electric field due to non-uniform distribution of electric field intensity at two sides. The electric field parameters output by the function generator are respectively 0V; 2.5V, 100 KHz; 5V, 100 KHz; and 0V; 3V, 100 KHZ; 6V and 100KHz, and the waveform is unified into square waves. The results of the experiment are shown at A, B, C, D, E, F in FIG. 5.
Example 3
By using the microfluidic chip 300 provided in example 1 and the method provided in example 2, the polystyrene microsphere sample was replaced with the circulating tumor cell H446, the circulating tumor cell was washed with PBS (containing 0.05% Tween), shaken and centrifuged twice, and then resuspended in the DEP buffer (8.5% sucrose [ wt/vol ], 0.3% glucose [ wt/vol ]) prepared by us, the conductivity was adjusted to 10ms/m, the electric field parameters output by the function generator were 6V,100KHz, and the waveforms were unified into square waves. The effect of capturing the circulating tumor cells H446 after the electrodes are activated is shown in FIG. 6, and the effect of simulating the potential electric field distribution in the dielectrophoresis region is shown in FIG. 7.
The above embodiments are merely preferred embodiments of the present invention, which are not intended to limit the scope of the present invention, and various changes may be made in the above embodiments of the present invention. All simple and equivalent changes and modifications made according to the claims and the content of the specification of the present application fall within the scope of the claims of the present patent application. The invention has not been described in detail in order to avoid obscuring the invention.
Claims (2)
1. A microfluidic device for manipulating capture of microparticles and cells based on non-contact dielectrophoretic forces, comprising: a sample injection pump, a micro-injector, a micro-fluidic chip and a waste liquid collector which are connected in sequence; the microfluidic chip is characterized in that the microfluidic chip is formed by laminating a glass substrate layer and a polydimethylsiloxane chip layer, wherein the polydimethylsiloxane chip layer comprises:
the sample injection port is connected with a sample injection channel;
the sheath flow sample inlet is led out from the sheath flow sample inlet and is divided into two sheath flow sample inlet channels, and the tail ends of the two sheath flow sample inlet channels are respectively converged with the sample inlet channels at two sides of the sample inlet channel to form a sample separation channel;
first and second liquid-electrode channels each independently in a closed loop shape disposed on both sides of the sample separation channel, the first liquid-electrode channel comprising: first electrode liquid introduction port and the most advanced wave structure of channel, the second liquid electrode channel includes: the second electrode liquid injection port and the channel tip gentle structure are respectively arranged on two opposite sides of the sample separation channel to apply a high-frequency high-voltage non-uniform electric field to the sample separation channel, the channel tip wavy structure and the channel tip gentle structure are respectively separated from the sample separation channel by solidified PDMS (polydimethylsiloxane), the channel tip wavy structure is a section of channel with a wavy shape, and the channel tip gentle structure is a section of channel with a linear shape;
the sample separation channel is led out from the tail end of the sample separation channel and is divided into a target product collection channel and a waste liquid collection channel which are formed in two parts respectively;
and a sample outlet connected with the target product collecting channel and the waste liquid collecting channel respectively;
the microfluidic device also comprises a plurality of target product liquid storage cavities connected with the target product collecting channel, and a step-by-step flow distribution structure is arranged between the target product collecting channel and the target product liquid storage cavities;
the micro-fluidic device also comprises electrodes respectively inserted into the first electrode liquid sample inlet and the second electrode liquid sample inlet, a high-voltage amplifier in signal connection with the electrodes, a function generator and a computer;
the sample feeding channel is arranged in the center of a snake-shaped channel, and the sheath flow sample feeding channel is respectively arranged at two sides far away from the sample feeding channel and then gradually approaches to converge with the sample feeding channel to form a sheath flow structure; before merging, the widths of the sample feeding channel and the sheath flow feeding channel are gradually narrowed; the width of the sample feeding channel is 200 μm, the width gradually narrows at the sheath flow structure part, the final width becomes 40 μm, the width of the sheath flow feeding channel is 100 μm, the width gradually narrows at the sheath flow structure part, and the final width becomes 30 μm;
the heights of the whole microfluidic chips are unified to be 40 mu m;
the first liquid electrode channel further comprises: first electrode liquid storage district and first electrode liquid buffer, the second liquid electrode channel still includes: a second electrode solution storage area and a second electrode solution buffer area.
2. A method for manipulating and capturing microparticles and cells based on non-contact dielectrophoresis force, comprising the steps of:
1) providing a microfluidic device for trapping microparticles and cells based on non-contact dielectrophoretic force manipulation according to claim 1;
2) placing the microfluidic chip in a vacuum pan for vacuumizing, then sucking DEP buffer solution by using a gun head and respectively injecting the DEP buffer solution into a first electrode solution injection port and a second electrode solution injection port, namely, performing a sample injection process on the DEP buffer solution through a sample injection port and a sheath solution injection port to ensure that the DEP buffer solution is filled in all channels to play a role in flushing and infiltration, facilitating the sample injection of a subsequent sample, simultaneously introducing sheath flow liquid and the sample into the microfluidic chip, adjusting a sample injection pump to ensure that the flow rate of the sheath flow liquid and the sample introduced into the microfluidic chip is 10mL/h-30mL/h, and opening an external circuit switch to control and capture microparticles or cells after stable sheath flow liquid in the microfluidic chip is formed; and
3) and observing the movement of the microparticles and the cells in the electric field in real time under a microscope, and analyzing the optimal physical parameters aiming at different microparticles or cells.
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| CN112280648A (en) * | 2020-09-30 | 2021-01-29 | 苏州莱博睿思生物科技有限公司 | Method for separating cells by using cell separation device |
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