CN112175824B - Full-automatic single cell capturing chip based on digital microfluidic technology and application thereof - Google Patents
Full-automatic single cell capturing chip based on digital microfluidic technology and application thereof Download PDFInfo
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Abstract
The invention discloses a full-automatic selective single cell capturing device based on a digital microfluidic technology and application thereof. The single cell capturing device can realize full-automatic single cell selective capturing, the capturing success rate is 100%, and the obtained single cells are subjected to morphological and molecular biological classification and identification, have high specificity and high sensitivity, and are suitable for completing single cell separation and subsequent application of circulating tumor cells in blood.
Description
Technical Field
The invention belongs to the technical field of digital micro-fluidic, and particularly relates to a full-automatic single particle/single cell capture chip based on a digital micro-fluidic technology and application thereof.
Background
The cell is a basic unit for the composition and function of a biological structure, and the cell analysis can provide an important theoretical basis for physiological processes such as metabolism, signal conduction and the like. Most of the traditional cell analysis is based on the population cells, and the analysis result is the result of the average of a large number of cells, and is an average data of the population state. At present, more and more researches show that the heterogeneity exists between cell individuals, a group of cells which are similar to each other are possible to have substantial differences inside, and the differences exist in the aspects of genome sequences, enzyme activities, protein expression and the like. These differences, reflected at the cellular level, manifest as heterogeneity of cellular function that is not negligible in basic and clinical medical research. In addition, in the process of cancer occurrence, due to certain contingencies of events such as gene mutation, cancer tissues have great heterogeneity, and the heterogeneity can also become one of the main barriers of clinical cancer treatment. For example: mutations in the Epidermal Growth Factor Receptor (EGFR) assist in diagnosis and are believed to have a critical role in the treatment of tyrosine kinase inhibitors targeted cancers. Efficient recognition of small numbers of EGFR-mutant cells is an urgent need for current clinical treatments. However, these mutant cell signals are often masked by the noise signals of most normal cells, preventing efficient identification of the mutant cells. Therefore, in recent years, scientists have been interested in a great deal of research on single cells, and hopefully, the essence of organisms can be better understood through the intensive research on these basic units participating in the in vivo reaction of organisms.
Although single cell analysis techniques are critical in many applications, the small size of single cells, low content of measured components, and biased amplification make the isolation and analysis of single cells challenging. Moreover, when the number of cells is large, a large number of repeated operations are required, which is time-consuming, and thus the experimental throughput cannot be increased.
The micro-fluidic chip utilizes micro-channels with different structures and external force fields with various forms to manipulate, process and control trace fluids or samples on a micro scale, thereby realizing the integration of partial or even all functions of the traditional laboratory on a microchip. Compared with the traditional method, the method has the advantages of low cost, high analysis speed, low reagent consumption and the like, and represents the development direction of miniaturization of biological and chemical analysis in the future. Therefore, the microfluidic chip is widely applied to the fields of neuroscience, stem cell biology, developmental biology, cancer diagnosis, personalized drug screening and the like as one of the most main research means for single cell separation and analysis. However, the limitations of the conventional microfluidic chip are also very obvious, and the conventional microfluidic chip needs a mechanical pump and a valve to be used in a matching manner, so that the integration difficulty is high, and the continuous multi-step processing of a sample is difficult to realize; the accurate control of the positions and the reaction time of the multiple reagents is realized; and single cell capture of a small amount of rare cells cannot be realized, and the method is especially ineffective in the field of high-efficiency high-throughput analysis of a large amount of single cells.
In order to solve the problems, a full-automatic single cell capture chip based on a digital microfluidic technology is provided. The chip can capture single cells automatically, and the capture efficiency of the single cells is high.
Disclosure of Invention
The invention aims to provide a full-automatic single cell capturing chip based on a digital microfluidic technology, which can capture single cells in a full-automatic and high-efficiency manner.
In order to achieve the purpose, the invention adopts the following technical scheme:
a full-automatic selective single cell capturing device based on a digital microfluidic technology comprises a digital microfluidic chip, an imaging system positioned above the digital microfluidic chip and a control circuit respectively connected with the digital microfluidic chip and the imaging system.
The digital microfluidic chip comprises an upper polar plate and a lower polar plate, wherein the lower polar plate comprises a substrate, an electrode layer, a dielectric layer and a locally hydrophilic hydrophobic layer, the upper polar plate is a hydrophobic ground electrode, the upper polar plate and the lower polar plate are parallel and opposite and are separated by a gap layer; the electrode layer comprises an electrode access port, a liquid storage pool electrode unit, a liquid drop generation channel electrode array, a single-particle/single-cell capture electrode array and a reaction electrode region, the liquid drop generation channel electrode array is respectively connected with the liquid storage pool electrode unit one by one, and the liquid drop generation channel electrode array and the liquid storage pool electrode unit are symmetrically arranged on two sides of the liquid drop generation channel electrode array by taking the single-particle/single-cell capture electrode array and the reaction electrode region as centers;
the locally-hydrophilized hydrophobic layer is a hydrophobic layer with locally-hydrophilized sites, and can be aligned to form hydrophilized capture sites in the center of a designated electrode, so that single cells can be captured by the adhesion of a hydrophilic structure;
the imaging system is positioned right above the digital microfluidic chip and used for acquiring image information at the hydrophilic capture site in the imaging area. After the liquid drops reach a hydrophilic capture position detection point, after two continuous image identifications of a bright field and a fluorescent field, outputting image information to enter a control circuit to be compared with a set image threshold value, outputting a corresponding control instruction by the control circuit according to a comparison result, and controlling an electrode driving circuit to output a voltage program with a corresponding change rule by the control instruction, thereby realizing the judgment of the success or failure of single cell capture;
the control circuit is respectively connected with the digital microfluidic chip and the imaging system and is used for respectively outputting a digital microfluidic chip control program and an imaging system operation program.
In a preferred embodiment of the invention, the electrode layer comprises 96 electrode access ports, eight reservoir electrode units, eight droplet generation channel electrode arrays, 10 single particle/single cell trapping electrode arrays and a reaction electrode region.
In a preferred embodiment of the present invention, the droplet generation channel electrode arrays are connected to the reservoir electrode units one by one, and are symmetrically arranged on both sides of the single-particle/single-cell trapping electrode array and the reaction electrode area with the center being the center.
In a preferred embodiment of the present invention, the shape of the hydrophilic capture sites in the hydrophobic layer includes a circle, a square, a half-moon, etc., and the size can be determined by the size of the electrode. Typically, the hydrophilic sites for capture may be circular in shape with a diameter of 50-500 microns, such as 100 microns, 150 microns, 200 microns, 250 microns, 300 microns, 350 microns, or 400 microns.
In a preferred embodiment of the invention, the particles may have a diameter of 5-200 microns, such as 5 microns, 10 microns, 15 microns, 20 microns, 30 microns, 70 microns, 80 microns or 90 microns.
In a preferred embodiment of the invention, the diameter of the cells may be 5-100 microns, such as 5 microns, 10 microns, 15 microns, 20 microns, 30 microns, 70 microns, 80 microns or 90 microns.
The hydrophilic and hydrophobic layer used by the digital microfluidic chip is made of Teflon or polytetrafluoroethylene PTFE aqueous dispersion, and is constructed on a dielectric layer through a local stripping technology to form a locally hydrophilic capture site.
To prevent evaporation of the droplets, the droplets are coated with an oil shell or immersed in an oil phase.
The reservoir electrode unit can achieve variation in droplet volume from 1 nanoliter to 10 microliters for different single particle/single cell capture systems.
In order to ensure the movement of the droplets in the single-particle/single-cell analysis system, surfactants are added to the droplets to prevent bioadhesion.
To selectively capture single particles/cells, image recognition and fully automated process selective capture are combined.
In a preferred embodiment of the present invention, the basic workflow of the digital microfluidic chip is as follows:
step A, placing the liquid with or without fluorescent cells in a liquid storage pool of a digital microfluidic chip, adding a proper amount of filling oil to wrap each liquid drop, pre-electrifying an electrode by using an integrated circuit, and turning to step B;
and B: controlling an electrode driving circuit by using a control circuit of an integrated circuit, and performing on-off control on electrodes according to a certain sequence, so that cell-containing liquid drops with a proper volume are generated from a liquid storage pool electrode unit, moving the liquid drops to the single cell capturing electrode by a channel electrode array connected with the liquid drops, repeating the process to enable each capturing electrode to load the cell-containing liquid drops, and turning to the step C;
and C: the control circuit of the integrated circuit controls the electrode driving circuit to enable liquid drops to pass through the single cell capture electrode array area, when single cells enter capture electrodes with hydrophilic structures, the electrodes of the single cell capture electrode array area are powered off, the liquid drops are settled on the surface of the chip under the action of gravity after standing for 1 minute, then the liquid drops are moved to the next electrode under the driving of the electrodes, and then the step D is carried out;
step D: e, shooting by using a bright field and a fluorescent field in sequence, outputting image information, and turning to the step E;
step E: and after image recognition is carried out on the bright field and the fluorescence field for two times continuously, image information is output to enter a control circuit to be compared with a set image threshold value, the control circuit outputs a corresponding control instruction according to a comparison result, the control instruction controls an electrode driving circuit to output a voltage program with a corresponding change rule, so that the judgment on whether the single cell capture is successful or not is realized, if the single cell capture is not a single particle/cell or not a target particle/cell, the control instruction repeats the step C, if the single cell capture is a target single particle/cell, the single cell capture is selectively completed, and the step F is carried out.
Compared with the prior art, the invention has the advantages that:
(1) the digital microfluidic chip comprises a digital microfluidic chip with a multilayer pattern and an integrated circuit. The digital microfluidic chip with the multilayer patterns consists of an upper polar plate and a lower polar plate, wherein the lower polar plate comprises a substrate, an electrode layer, a dielectric layer and a locally hydrophilic hydrophobic layer, and the upper polar plate comprises a hydrophobic ground electrode; the integrated circuit is a digital microfluidic circuit control system. The device has the advantages of full automation, easy integration, addressable property, small volume, less reagent consumption and cost saving;
(2) the single cell capturing device can capture single cells with 100% selectivity;
(3) the method has the advantages that the method has no cell loss basically, can enable all capture sites to capture single cells under the condition of limited cell number, can control the capture number of the single cells according to the number of the capture sites of the chip, and is particularly suitable for analysis of rare samples, such as analysis of Circulating Tumor Cells (CTC), stem cells and other rare cells;
(4) the invention can realize more direct and greater space-time control on the generated discrete micro-droplets, and has advantages on full-automatic processing and full integration.
Drawings
Fig. 1 is a schematic diagram of the overall structure of the digital microfluidic chip of the present invention.
FIG. 2 is a schematic diagram of a single cell selective capture process according to the present invention.
FIG. 3 is a schematic diagram of the effect of single cell selective capture according to the present invention.
FIG. 4 is a graph showing the capture efficiency of single cell selective capture under different mixed cell ratios (blue fluorescence: green fluorescence) according to the present invention.
Detailed Description
The present invention will be described in detail below with reference to the accompanying drawings, which take the example of single cell whole genome amplification on a digital microfluidic chip.
Referring to fig. 1, the single-cell capturing device includes a digital microfluidic chip 1, an imaging system located above the digital microfluidic chip, and a control circuit respectively connecting the digital microfluidic chip and the imaging system.
The digital microfluidic chip comprises an upper polar plate and a lower polar plate, wherein the upper polar plate sequentially comprises a hydrophobic layer, a grounding conductive layer and an upper polar plate substrate from bottom to top;
the lower plate comprises a glass substrate 1 (in other embodiments, the lower plate can also be made of other materials), an electrode layer, a dielectric layer and a locally-hydrophilized hydrophobic layer, wherein the electrode layer is manufactured on the surface of the glass substrate by a wet etching technology (in other embodiments, methods such as a circuit board printing technology and a printing technology can also be used), the dielectric layer is uniformly coated on the upper surface of the electrode layer, the locally-hydrophilized hydrophobic layer is used for capturing single particles/single cells, a specific hydrophilic pattern is formed on the surface of the dielectric layer by a local stripping technology and the like, and a hydrophobic pattern is formed in the rest area of the dielectric layer;
the electrode layer is a chromium electrode array 2 (in other embodiments, other conductive metals are also possible) with a specific pattern, and comprises six reservoir electrode units 4, six droplet generation channel electrode arrays 5, ten single-particle/single-cell capture electrode arrays and reaction electrode regions 6 (in fig. 1, 3 is a hydrophilic site, and the single-particle/single-cell capture electrode arrays and the reaction electrode regions 6 are located below the hydrophilic site 3), the droplet generation channel electrode arrays 5 are respectively connected with the reservoir electrode units 4 one by one, and are symmetrically arranged on two sides of the reservoir electrode units (in other embodiments, other numbers and other shapes of electrode arrays are also possible) by taking the single-particle/single-cell capture electrode arrays and the reaction electrode regions as centers;
the locally hydrophilized hydrophobic layer is a hydrophobic layer formed by a local lift-off technique with locally hydrophilized sites that are aligned to form circular shaped hydrophilized capture sites (in other embodiments, other shapes of hydrophilized capture sites are also possible) at the center of a given electrode.
The imaging system is positioned right above the digital microfluidic chip and used for acquiring image information at the hydrophilic capture site in the imaging area. After the liquid drops reach a hydrophilic capture position detection point, after two continuous image identifications of a bright field and a fluorescent field, outputting image information to enter a control circuit to be compared with a set image threshold value, outputting a corresponding control instruction by the control circuit according to a comparison result, and controlling an electrode driving circuit to output a voltage program with a corresponding change rule by the control instruction, thereby realizing the judgment of the success or failure of single cell capture;
the control circuit is respectively connected with the digital microfluidic chip and the imaging system and is used for respectively outputting a digital microfluidic chip control program and an imaging system operation program.
The digital microfluidic manufacturing process comprises the following steps:
manufacturing a lower polar plate:
(a) evaporating a 300-nanometer thick chromium layer on a lower plate glass substrate by using a magnetron sputtering method, and forming a chromium electrode array with a specific structure by wet etching;
(b) the dielectric layer material is an insulating material with high dielectric constant, uniform thickness and strong breakdown resistance, and photoresist is formed in a spin coating mode in the embodiment;
(c) the hydrophobic layer is made of teflon, and in this embodiment, the hydrophobic layer is partially peeled off by using a polytetrafluoroethylene PTFE aqueous dispersion through a local peeling and alignment technique, so that a hydrophilic surface below is exposed, and a locally hydrophilized site is formed in the center of a designated electrode. In other embodiments, the locally hydrophilized sites can also be formed by other existing means.
Manufacturing an upper polar plate:
(a) the upper polar plate substrate is made of any insulating transparent material, such as glass;
(b) the grounding conductive layer is made of materials with high light transmittance and good visibility, such as ITO and the like, and is formed by deposition;
(c) the hydrophobic material of the upper plate is generally identical to the hydrophobic material of the lower plate, and is typically Teflon, and in this embodiment is formed by a spin-on annealing process using an aqueous dispersion of Polytetrafluoroethylene (PTFE).
The upper and lower polar plates are separated by a gap layer with a certain thickness, a sandwich structure is formed by the upper and lower polar plates and liquid drops in the upper and lower polar plates, and the liquid drops are led in through automatic sample introduction through holes corresponding to the positions of the upper polar plate above the liquid storage pool electrode of the lower polar plate. After the sample introduction is finished, the digital microfluidic chip is subjected to oil sealing, so that the effects of isolating pollution and promoting liquid drop movement are achieved.
As a preferred embodiment of the invention, the cells selected are EGFR expressing H1975 cells, the cell diameter being 10-20 microns.
As a preferred embodiment of the invention, the specific working process of the invention is as follows:
step A: loading each reagent in a liquid storage pool electrode unit of the digital microfluidic chip respectively, adding a proper amount of filling oil to wrap each liquid drop, pre-electrifying the electrodes by using an integrated circuit, and turning to the step B;
and B: controlling an electrode driving circuit by using a control circuit of an integrated circuit, performing on-off control on electrodes according to a certain sequence, enabling liquid drops to be broken under the traction of the electrodes so as to generate cell-containing liquid drops with a proper volume from an electrode unit of a liquid storage pool, moving the liquid drops to a single-cell capturing electrode by a channel electrode array connected with the liquid drops, repeating the process to enable each capturing electrode to load the cell-containing liquid drops, and turning to the step C;
and C: the control circuit of the integrated circuit controls the electrode driving circuit to enable liquid drops to pass through the single cell capture electrode array area, when single cells enter capture electrodes with hydrophilic structures, the electrodes of the single cell capture electrode array area are powered off, the liquid drops are settled on the surface of a chip under the action of gravity after standing for 1 minute, then the liquid drops are moved to the next electrode under the driving of the electrodes, whether the hydrophilic areas are the single cells or not is judged by combining image recognition, and if the hydrophilic areas are the single cells, the capture is finished; if the number of particles/cells is multiple or no particles/cells, the operation is repeated, so that the capture of multiple single cells on the chip can be realized, and then the step D is carried out.
Step D: for rare samples, driving the removed droplets back into the reservoir electrodes can achieve nearly lossless capture; for normal samples, the removed droplets are directly driven into a waste reservoir, and step E is carried out.
Step E: and (3) leading out cell lysate (0.2 percent Triton X-100) from the other liquid storage pool electrode unit by the control electrode driving circuit, driving the cell lysate to the single cell capturing structure to crack the cells, releasing cell contents contained in the cell lysate into the liquid drop after the cells are cracked, and transferring to the step F.
Step F: and controlling an electrode driving circuit to lead out the single cell whole genome amplification reagent from the next liquid storage pool electrode unit, mixing the reaction processes in a liquid drop driving mode, and taking out the liquid drops after the reaction is finished to perform subsequent analysis and research.
Example 1:
mixing the two kinds of cells dyed with blue fluorescence and green fluorescence according to a certain proportion, and placing the mixture into a liquid storage pool electrode unit of a digital microfluidic chip. An array of 10 hydrophilic micropores with an aperture of 200 μm was arranged on the lower plate of the chip. The cell drop is passed into the chip and driven to the hydrophilic site area for cell loading, and the specific fluorescence stained single cell enters the hydrophilic site through the dynamic regulation of the fluid, while other cells do not enter the area. And pulling the cell away from the hydrophilic site, and generating a hydrophilic droplet containing single cells through negative generation to realize the single cell separation of the specific cell (figure 2).
By the above selective capturing operation, green fluorescence-stained cells were all captured in the first row and blue fluorescence-stained cells were all captured in the second row of the 10 hydrophilic microwell arrays, as shown in fig. 3.
The mixing ratio of the two different stained cells was adjusted, the above single cell selective capture operation was continuously repeated, and the target cell capture efficiency at different purities was calculated, to obtain fig. 4. Experiments prove that the high-efficiency target cell capture can be still realized under the condition of extremely low purity.
The applicant declares that the present invention is described by the above embodiments as the detailed features and the detailed methods of the present invention, but the present invention is not limited to the above detailed features and the detailed methods, that is, it is not meant that the present invention must be implemented by relying on the above detailed features and the detailed methods. It will be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of selected components, selection of specific modes, etc., are intended to be within the scope and disclosure of the present invention.
Claims (9)
1. A full-automatic selective unicell capture device based on digital microfluidic technology which characterized in that: the device comprises a digital microfluidic chip, an imaging system positioned above the digital microfluidic chip and a control circuit respectively connected with the digital microfluidic chip and the imaging system;
the digital microfluidic chip comprises an upper polar plate and a lower polar plate, wherein the lower polar plate comprises a substrate, an electrode layer, a dielectric layer and a locally hydrophilic hydrophobic layer, the upper polar plate is a hydrophobic ground electrode, the upper polar plate and the lower polar plate are parallel and opposite and are separated by a gap layer; the electrode layer comprises an electrode access port, a liquid storage pool electrode unit, a liquid drop generation channel electrode array, a single particle/single cell capture electrode array and a reaction electrode area, and the liquid drop generation channel electrode array is respectively connected with the liquid storage pool electrode unit one by one;
the locally-hydrophilized hydrophobic layer is a hydrophobic layer with locally-hydrophilized sites, and can be aligned to form hydrophilized capture sites in the center of a designated electrode, so that single cells can be captured by the adhesion of a hydrophilic structure;
the imaging system is positioned right above the digital microfluidic chip and is used for acquiring image information at a hydrophilic capture site in the imaging area; after the liquid drops reach a hydrophilic capture position detection point, after two continuous image identifications of a bright field and a fluorescent field, outputting image information to enter a control circuit to be compared with a set image threshold value, controlling the circuit to output a corresponding control instruction according to a comparison result, and controlling an electrode driving circuit to output a voltage program with a corresponding change rule by the control instruction, thereby realizing the judgment of the success or failure of single cell capture;
the control circuit is respectively connected with the digital microfluidic chip and the imaging system and is used for respectively outputting a digital microfluidic chip control program and an imaging system operation program.
2. The fully automatic selective single-cell capture device based on digital microfluidic technology according to claim 1, characterized in that: the electrode layer comprises 96 electrode access ports, eight reservoir electrode units, eight droplet generation channel electrode arrays, 10 single particle/single cell trapping electrode arrays and a reaction electrode region.
3. The fully automatic selective single-cell capture device based on digital microfluidic technology according to claim 1, characterized in that: the droplet generation channel electrode arrays are respectively connected with the liquid storage pool electrode units one by one, and are symmetrically arranged on two sides of the single-particle/single-cell capture electrode array and the reaction electrode area by taking the single-particle/single-cell capture electrode array and the reaction electrode area as centers.
4. The fully automatic selective single-cell capture device based on digital microfluidic technology according to claim 1, characterized in that: the locally hydrophilized hydrophobic layer is formed by a local peeling technique.
5. The fully automatic selective single-cell capture device based on digital microfluidic technology according to claim 1, characterized in that: the diameter of the hydrophilization capture site is 50-500 microns.
6. The fully automatic selective single-cell capture device based on digital microfluidic technology according to claim 1, characterized in that: the reservoir electrode unit is capable of achieving droplet volumes varying from 1nL to 10 μ L for different single particle/single cell capture systems.
7. The fully automatic selective single-cell capture device based on digital microfluidic technology according to claim 1, characterized in that: the upper polar plate adopts a transparent conductive film.
8. A full-automatic selective single cell capture method based on digital microfluidic technology, using the device of any one of claims 1 to 7, comprising the following steps:
step A, placing the liquid with or without fluorescent cells in a liquid storage pool of a digital microfluidic chip, adding a proper amount of filling oil to wrap each liquid drop, pre-electrifying the electrodes by using an integrated circuit, and turning to step B;
and B: controlling an electrode driving circuit by using a control circuit of an integrated circuit, and performing on-off control on electrodes according to a certain sequence, so that cell-containing liquid drops with a proper volume are generated from a liquid storage pool electrode unit, moving the liquid drops to a single-particle/single-cell capturing electrode by a channel electrode array connected with the liquid drops, repeating the process to enable each capturing electrode to load the particle-containing/cell-containing liquid drops, and turning to the step C;
and C: the control circuit of the integrated circuit controls the driving of the electrodes to enable liquid drops to pass through the single-cell capture electrode array area, when cells enter the capture electrodes with hydrophilic structures, the electrodes of the single-cell capture electrode array area are powered off, after the liquid drops are settled, the particles/cells are settled on the surface of the chip under the action of gravity, then the liquid drops are driven by the electrodes to move to the next electrode, and then the step D is carried out;
step D: e, shooting by using a bright field and a fluorescent field in sequence, outputting image information, and turning to the step E;
step E: and after two continuous image identifications, outputting image information, entering a control circuit, comparing the image information with a set image threshold, outputting a corresponding control instruction by the control circuit according to a comparison result, controlling an electrode driving circuit to output a voltage program with a corresponding change rule by the control instruction, thereby realizing the judgment of success or failure of single cell capture, continuously repeating the step C by the control instruction if the single cell is not a single particle/cell or a target cell, and selectively capturing the single cell if the single cell is the target single cell.
9. The fully automatic selective single cell capture method based on digital microfluidic technology according to claim 8, characterized in that: in step A, surfactants are added to the liquid with and without fluorescent cells to prevent bioadhesion and to facilitate droplet movement in a single particle/single cell assay system.
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| CN114308152A (en) * | 2021-12-13 | 2022-04-12 | 中国科学院上海微系统与信息技术研究所 | Digital microfluidic chip and preparation method and application thereof |
| CN114438012A (en) * | 2022-01-26 | 2022-05-06 | 合肥工业大学 | Flexible capture method for micron-sized particles or cells |
| CN114632564B (en) * | 2022-04-20 | 2024-03-08 | 香港城市大学深圳研究院 | Integrated micro-fluidic chip and primary circulating tumor cell in-vitro treatment method |
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