WO2017177839A1 - Puce de réseau de micro-cuvettes super-hydrophobes, son procédé de préparation et ses applications - Google Patents

Puce de réseau de micro-cuvettes super-hydrophobes, son procédé de préparation et ses applications Download PDF

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WO2017177839A1
WO2017177839A1 PCT/CN2017/078946 CN2017078946W WO2017177839A1 WO 2017177839 A1 WO2017177839 A1 WO 2017177839A1 CN 2017078946 W CN2017078946 W CN 2017078946W WO 2017177839 A1 WO2017177839 A1 WO 2017177839A1
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micro
pit
layer
pit array
array
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Chinese (zh)
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刘鹏
张鹏飞
张健雄
边升太
程一淳
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清华大学
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    • C—CHEMISTRY; METALLURGY
    • 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
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/12—Well or multiwell plates
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/20—Material Coatings
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0684—Cells of the urinary tract or kidneys
    • C12N5/0686—Kidney cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/069—Vascular Endothelial cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0693—Tumour cells; Cancer cells
    • C12N5/0694—Cells of blood, e.g. leukemia cells, myeloma cells

Definitions

  • the invention relates to a super-hydrophobic micro-pit array chip and a preparation method and application thereof.
  • Cell microarray is an array of tiny cells formed by chemical and physical separation. Such cell microarrays are gaining more and more attention in biological research because of their high flux, low reagent consumption, and low cost. Chemically separated cells are mostly formed by surface modification, by making differential modifications to the surface, so that cells can only grow at specific array positions to form a cell dot matrix. The physical separation can be performed by forming a micro-pit, blocking the cells, and more by injection molding, or by soft lithography. There are also cell microarray platforms that are simultaneously separated by chemical and physical methods.
  • Cellular microarrays and cell microarray platforms that implement cell microarrays play an important role in high-throughput drug screening, cell transfection, and stem cell differentiation.
  • High-throughput cell microarrays provide a good platform for studying the association between cellular genes and phenotypes and exploring the functions of genes. High-throughput reverse-transfection and lentiviral transcription studies have demonstrated the enormous potential of cell microarrays in gene function studies.
  • High-throughput cell microarrays provide an important platform for exploring the influencing factors of stem cell differentiation and exploring the microenvironment of stem cell differentiation.
  • High-throughput cell microarrays have a distinct advantage in screening cells that secrete monoclonal antibodies directed against specific antigens. High-throughput cell microarrays can be compared to traditional limiting dilution methods. Significantly improve the screening efficiency, which is of great significance for the study of cell secretions.
  • the random optical reconstruction microscope is one of the highest resolution optical microscopes.
  • Ultra-high resolution imaging is of great significance for the study of biology. Many subcellular structures are on the micron to nanometer scale, and the existence of diffraction limits limits our observation of these biological samples using optical microscopy. For example, the skeletal protein microfilaments of the cells are very dense. Under the fluorescence microscope, the image is very blurred and the details cannot be seen. The resolution of the electron microscope can reach about 1 nm, which clearly shows the details of the cytoskeleton. However, electron microscopy can hardly make live samples, and the specificity is not as good as fluorescence microscopy.
  • Ultra-high-resolution fluorescence microscopy technology has gradually matured, but the operation steps are often tedious and cumbersome, and the detection efficiency is not high. Therefore, high-throughput ultra-high resolution imaging using high-throughput cell microarrays accelerates research in this field. Development is very important.
  • the high-throughput cell microarray experimental platform is a research platform of great value, and it has very important value and significance in both basic research and practical application.
  • microfluidic technology has accumulated a technical foundation for the development of new low-cost cell detection platforms.
  • a cell microarray can be formed, and different drug molecules can be specifically added to different cell lattices by controlling the switch of the pump valve, thereby achieving high-throughput drug analysis.
  • the construction of this microfluidic platform is relatively complicated and is not suitable for large-scale promotion in the field of biological research.
  • PDMS micropores, PEG micropores or other micro-pits prepared by various materials have become the important cell detection and analysis platform due to their simple preparation method and low cost. But since all the micropores are immersed in the same medium, the microenvironment of the cell array It cannot be controlled independently. For example, in high-throughput drug screening, water-soluble molecules will dissolve and diffuse, making it difficult to avoid cross-contamination and limiting the use of such platforms. Therefore, many researchers are working to solve this problem, but the results are not ideal, because it will cause other problems while solving the cross-contamination problem. For example, some researchers have used oil seals to isolate micro-pits, although cross-contamination can be solved to some extent, but at the same time it brings problems such as inconvenient operation.
  • microdevices currently developed for high-throughput cell culture, detection, and analysis are often poorly biocompatible, failing to ensure normal cell growth, and limiting these microdevices in high-throughput cell biology experiments.
  • Application in . the doubling time of cells cultured on microdevices is often much greater than the rate of cell proliferation in multiwell plates; after a period of incubation, cells on microdevices have abnormal cell phenotypes, such as the inability to express specific proteins of the cells. and many more. Therefore, most micro-devices cannot carry out the cultivation and analysis of primary cells and stem cells that are very sensitive to the environment, and the high-throughput microarrays of primary cells and stem cells are very important in the research and exploration of personalized medicine and regenerative medicine. Significance.
  • STORM imaging is extremely complex in the processing of samples.
  • the independent environment in each micro-pit can be individually controlled for large-scale STORM sample preparation, compared to traditional STORM sample preparation, the efficiency is significantly improved, greatly improving the information density of super-resolution imaging and reducing the experimental cost.
  • high-resolution imaging of multiple samples was performed by sample-by-sample processing, which was time-consuming, labor-intensive, and inefficient, which greatly limited the high. Resolve the development of imaging.
  • the object of the present invention is to provide a super-hydrophobic micro-pit array chip and a preparation method and application thereof.
  • the super-hydrophobic ultra-micro pit array chip is low in cost, simple in operation, and can well avoid crossover under the condition of ensuring normal growth state of cells. Contamination, while ensuring good biocompatibility, greatly reduces the cost of high-throughput cell analysis, and further promotes the use of high-throughput cell detection technology, in high-throughput drug screening, high-throughput high-throughput It plays an important role in applications such as resolution imaging.
  • the invention provides a micro-pit array chip which comprises a micro-pit array layer, and a surface of the micro-pit array layer on which a micro-pit or a micro-pit bottom is provided is a super-hydrophobic surface.
  • the superhydrophobic surface in the present invention means a surface having a contact angle with water (or an aqueous solution) of more than 150° and a rolling angle of less than 10°.
  • the micropits include the bottom of the dimple and the entire sidewall of the dimple.
  • the surface other than the micro-pit is a super-hydrophobic surface means that the surface between the micro-pit and the micro-pit is a super-hydrophobic surface (the bottom surface and the side surface of the micro-pit are hydrophilic).
  • the surface other than the bottom of the micropit is a superhydrophobic surface, which means that the side surface of each micropit and the surface between the micropit and the micropit are superhydrophobic surfaces (the bottom surface of the micropit is hydrophilic
  • the micro-pit array chip of the invention can automatically form a micro-droplet array of an aqueous solution due to its special super-hydrophobic modification, thereby ensuring physical isolation of the micro-array and avoiding cross-contamination between the arrays.
  • the shape, size, depth, processing mode, and the like of the micro-pit array layer are not limited.
  • the experimental throughput of the micro-pit array chip of the present invention is high.
  • more than 1000 droplet arrays can be constructed on a common slide size (76 mm * 26 mm) chip; reagent consumption Very small, each micropit has a volume of approximately 50nL, greatly reducing the cost of high-throughput detection and analysis.
  • the micro-pit array chip may be the following (A) or (B):
  • the surface other than the micro-pit is a super-hydrophobic surface
  • the micro-pit array chip includes a micro-pit array layer and a super-hydrophobic layer attached to the surface of the micro-pit array layer (the micro-pit array layer has a hydrophilic layer) Sex);
  • the surface other than the bottom of the micropit is a superhydrophobic surface
  • the micropit array layer comprising a base layer and a microporous array layer attached to the surface of the base layer, the microporous array layer being superhydrophobic Made of material (the base layer is hydrophilic).
  • the superhydrophobic layer in the micropit array chip (A), may have a thickness of 10 to 200 ⁇ m, preferably 100 to 150 ⁇ m, more preferably 100 ⁇ m.
  • the super-hydrophobic layer can be prepared from any material that can be made into a super-hydrophobic surface, and the material and preparation manner are not limited, for example, in the present invention.
  • the superhydrophobic layer can be prepared by the following method: a superhydrophobic prepolymer solution having the following composition: 24 wt% methyl propyl acrylate, 16 wt% ethylene glycol dimethacrylate, 60 wt. %1-sterol (1-decanol) and 1wt% 2,2-dimethoxy-2-phenylacetophenone are thoroughly mixed and injected into two silanized slides, which are exposed to UV light.
  • the superhydrophobic layer is obtained.
  • the method of bonding to the substrate layer includes, but is not limited to, the method of pasting described in the embodiments of the present invention.
  • materials of the micro-pit array layer include, but are not limited to, polydimethylsiloxane (PDMS) and polymethyl methacrylate (PMMA). , Polymethyl methacrylate), at least one of polycarbonate (PC, Polycarbonate), stainless steel, glass, and the like.
  • the micro-pit array layer in the micro-pit array chip (A), in order to meet the unique requirement of super-resolution imaging, the micro-pit array layer includes a base layer and a micro-hole array attached to the surface of the base layer
  • the layer may be an ultra-high resolution imaging-specific optical slide.
  • the ultra-high resolution imaging dedicated micro-pit array chip can meet the working distance of 100 times the objective lens in ultra-high resolution imaging, and can realize automated high-throughput imaging.
  • the micro-hole array layer can be made of any super-hydrophobic material, and the material and preparation manner are not limited, for example, in a specific embodiment of the present invention.
  • the microporous array layer can be prepared by the following method: a superhydrophobic prepolymer solution having the following composition: 24 wt% methyl propyl acrylate, 16 wt% ethylene glycol dimethacrylate, 60 wt% 1-decanol and 1wt% 2,2-dimethoxy-2-phenylacetophenone are thoroughly mixed and injected into the void of the microsilica array clamped microarray array, UV The microwell array layer is obtained by exposing the lamp.
  • the material for preparing the underlayer includes, but not limited to, polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), Polycarbonate (PC, Polycarbonate), stainless steel, glass, etc.
  • PDMS polydimethylsiloxane
  • PMMA polymethyl methacrylate
  • PC Polycarbonate
  • stainless steel glass, etc.
  • the present invention further provides a method for fabricating the above-described micro-pit array chip, which comprises the steps of forming a super-hydrophobic surface on a surface of the micro-pit array layer having a micro-pit on a surface other than a micro-pit or a pit. .
  • the method may be the following method 1) or method 2):
  • step 2-b) taking another substrate, aligning and fixing together with the array of protrusions in step 2-a); injecting a superhydrophobic prepolymer into the gap between the two substrates, and solidifying and separating,
  • the above-described micropit array chip having a surface other than the bottom of the micropit is a superhydrophobic surface.
  • the method for preparing the micro-pit array layer may include the following steps: in the micro-hole array layer The surface is adhered with a layer of glue and then attached to the surface of the substrate layer to obtain the ultra-high resolution imaging dedicated micro-pit array chip.
  • the method in the above manufacturing method, in the method 1), the method can realize fine modification up to 200 micrometers, the modification method can be applied to pattern modification of various shapes, and the modification method has almost no choice for the substrate micro-pit chip material. Sexuality can be applied to the superhydrophobic modification of micro-pit chips of various materials.
  • the shape of the protrusion array matches the shape of the micropit in the micropit array layer, in a specific embodiment of the invention
  • the array of protrusions may be an array of micro-pillars; the array of micro-pillars may specifically be an array of negative-adhesive micro-pillars, which are formed by exposure and development.
  • the present invention further provides a method for performing high-throughput cell culture using the above-described micro-pit array chip, which comprises at least one of the following steps a) to d):
  • a dosing step which includes the following steps:
  • the liquid adding step can be completed by dropping the cell culture solution dropwise above the micro-pit array chip
  • the cells are added one by one to complete the step of adding cells;
  • step of droplet culture which comprises the following steps:
  • the chip After the step of adding the cells, the chip is immersed in the cell culture solution, and then the excess culture solution of the immersed chip is removed to form an array of droplets, and the cells are cultured to realize the droplet culture;
  • the step of changing the liquid is completed by withdrawing the excess cell culture medium until the droplet array is reformed.
  • the droplet culture can be carried out under saturated air humidity.
  • the cells may be subjected to droplet culture by placing the micro-pit array chip in a set culture dish, and the set culture dish is structured as follows: a large culture dish is placed in a small culture dish. Outside, and add sterilized water between the large petri dish and the small culture.
  • the method may further comprise the step of knife coating the surface of the formed droplet array, in a specific embodiment of the invention
  • a glue stick can be used to gently sweep across the surface of the array of droplets in the microwell array chip.
  • FIG. 1 is a schematic structural view of a superhydrophobic micropit array chip in Embodiment 1. Each mark in Fig. 1 is as follows: 1 micro-pit array layer, 2 super-hydrophobic layer.
  • Example 2 is a flow chart of micro-grafting to prepare a super-hydrophobic micro-pit array chip in Example 1.
  • FIG. 3 is a photograph, a micro-pit SEM photograph and a contact angle photograph of the super-hydrophobic micro-pit array chip prepared in Example 1, wherein FIG. 3(a) is a photograph of a super-hydrophobic micro-pit array chip, and FIG. 3(b) is a photograph of FIG. Fig. 3(a) is a transverse SEM photograph in the boxed area, Fig. 3(c) is a photograph of the contact angle of the superhydrophobic layer in the boxed area in Fig. 3(b), and Fig. 3(d) is a square in Fig. 3(b) Longitudinal SEM photographs of the area.
  • Example 4 is a SEM photograph of a superhydrophobic micro-pit array chip of various shapes and sizes prepared in Example 1.
  • FIG. 5 is a schematic structural view of a super-hydrophobic micro-pit array chip in Embodiment 2.
  • FIG. Each mark in Figure 5 is as follows: 1 base layer, 2 superhydrophobic microwell array layer.
  • Example 6 is a flow chart of in situ synthesis of a superhydrophobic micropit array chip in Example 2.
  • Example 7 is a photograph of a superhydrophobic micropit array chip prepared in Example 2.
  • Embodiment 8 is a flow chart of preparing a super-high resolution imaging super-hydrophobic micro-pit array chip in Embodiment 3.
  • Figure 9 is a diagram showing the spontaneous formation of droplets in a superhydrophobic micropit array chip of the present invention.
  • Figure 10 is a schematic view of the surface of the ultra-hydrophobic chip droplet array using a glue stick and the experimental results of the droplet volume variance before and after the treatment, and the corresponding SEM photograph, wherein Figure 10 (a) is a lightly coated stick Schematic diagram of sweeping the surface of the superhydrophobic chip droplet array, Fig. 10(b) is the experimental result of the droplet volume variance before and after the treatment and the corresponding SEM photograph.
  • Figure 11 is a schematic diagram of high-throughput cell microarray culture and detection analysis using the superhydrophobic micro-pit array chip of the present invention.
  • FIG. 12 is a view showing a device for suppressing droplet evaporation used in high-throughput cell microarray culture using the superhydrophobic micro-pit array chip of the present invention, wherein FIG. 12a is a schematic structural view of a device for controlling humidity when the chip is cultured in an incubator.
  • Figure 12b is a physical photograph of Figure 12a.
  • FIG. 13 is an experimental result of droplet culture using the superhydrophobic micro-pit array chip of the present invention, wherein FIG. 13a is a culture of hamster kidney cell BHK-21, human umbilical vein epithelial cell HUVEC and human chronic myeloid leukemia cell K562, respectively. Photomicrographs after hours, 72 hours, and 120 hours, and micrographs after Calcein AM/PI staining, Figure 13 (b) for hamster kidney cells BHK-21 and human umbilical vein epithelial cells HUVEC for droplet culture And the survival rate after immersion culture, Fig. 13(c) shows hamster kidney cell BHK-21, human umbilical vein epithelial cell HUVEC, immersion culture, droplet culture, and cells cultured in a common 24-well plate. Proliferation rate.
  • Figure 14 is a photomicrograph of immunofluorescence staining and angiogenesis detection of HUVECs cultured for 72 hours using the superhydrophobic micro-pit array of the present invention, wherein 14a is the result of immunofluorescence staining of CD 31 protein, and 14b is VE-cadherin. As a result of protein immunofluorescence staining, 14c is the result of angiogenesis assay.
  • FIG. 15 is a result of high-throughput analysis and detection by adding different fluorescent dye molecules to the superhydrophobic micro-pit array of the present invention by a spray pattern technique, wherein FIG. 15a is a photograph after adding different fluorescent dye molecules, and FIG. 15b is a different addition.
  • the components of the superhydrophobic polymer prepolymer solution in the following examples include: 24 wt% butyl methacrylate (BMA), 16 wt% ethylene dimethacrylate (EDMA), 60 wt. %1-nonanol (1-decanol) and 1% by weight of 2,2-dimethoxy-2-phenylacetophenone (relative to the sum of the monomers) (2,2-dimethoxy-2-phenylacetophenone, DMPAP) (all purchased from Sigma-Aldrich).
  • BMA butyl methacrylate
  • EDMA 16 wt% ethylene dimethacrylate
  • DMPAP 2,2-dimethoxy-2-phenylacetophenone
  • Preparation method is to put on The raw materials of the ratio were thoroughly mixed on a homogenizer for 1 hour and then used.
  • the slides in the following examples were washed and alkylated before use.
  • the slides were washed three times with water and thoroughly purged with nitrogen.
  • the alkylation modification step is: immersing the above-mentioned washed slide with a 20% (by volume) 3-(trimethoxysilyl)propyl methacrylate ethanol solution
  • the silylation modification can be completed, and after 1 hour, it is washed three times with acetone, and dried under nitrogen for use.
  • FIG. 1 A schematic diagram of the structure of the super-hydrophobic micro-pit array chip is shown in FIG. 1. It comprises a micro-pit array layer 1 and a super-hydrophobic layer 2 attached to the surface of the micro-pit array layer, wherein the super-hydrophobic layer 2 has a thickness of 100 ⁇ m.
  • a micro-grafting technique is used to prepare a super-hydrophobic micro-pit array chip.
  • the specific steps are as follows:
  • PDMS micro-pit chip preparation using the processed silicon wafer SU-8 mold (manufactured by Boao Biochip Co., Ltd.), wrapped in tin foil and used as a container for the mold.
  • the PDMS prepolymer solution was prepared.
  • the volume ratio of monomer to catalyst was 10:1 (purchased from Dow Corning, catalog number SylgardR 184).
  • the glass rod was thoroughly stirred and mixed, and then placed in a vacuum oven for 30 minutes to remove bubbles in the solution. .
  • the prepolymer solution was poured onto the SU-8 mold, placed in an oven at 80 ° C for 2 h or more, and then removed from the mold for use.
  • the super-hydrophobic micro-pit array processing method based on micro-grafting technology in this embodiment can realize fine modification up to 200 micrometers, and the modification method can be applied to pattern modification of various shapes (as shown in FIG. 4), and this The modification method has almost no selectivity for the substrate micro-pit chip material, and can be applied to super-hydrophobic modification of various material micro-pit chips.
  • FIG. 5 The schematic diagram (side view) of the super-hydrophobic micro-pit array chip is shown in FIG. 5, which comprises a base layer 1 and a superhydrophobic microporous array layer 2 attached to the surface of the substrate (the micro-hole array layer 2 is made of a superhydrophobic material). ).
  • the superhydrophobic micro-pit array chip is prepared by in-situ synthesis technology, and the specific steps are as follows:
  • Negative gel microcolumn array preparation 100 ⁇ m thick negative gel was spread on the silanized slide, and developed to obtain a microcolumn array having a height of 100 ⁇ m, a diameter of 500 ⁇ m, and a center distance of 1000 ⁇ m.
  • FIG. 1 A photograph of the superhydrophobic micropit array chip prepared in this embodiment is shown in FIG.
  • Embodiment 3 Super-hydrophobic micro-pit array chip for ultra-high resolution imaging
  • This embodiment is based on the unique requirement of super-resolution imaging, and the preparation method of the micro-pit substrate array is adjusted accordingly.
  • the structure diagram of the super-hydrophobic micro-pit array chip is the same as that of FIG. 1 , including the micro-pit array layer 1 and the bonding in the micro-pit.
  • the superhydrophobic layer 2 on the surface of the array layer, only the micropit array layer 1 is composed of a base layer (an ultra-high resolution imaging-dedicated optical slide) and a microporous array layer attached to the substrate.
  • Photoresist dry film microarray preparation a microporous array film was obtained by a pre-bake, exposure, post-baking, developing process using a 100 ⁇ m thick photoresist dry film.
  • (h) Re-leveling Take Dow Corning 3140 as an example. After 30s of squeezing at 7000 rpm, a very thin layer of glue can be obtained on the PMMA layer. A thin layer of thin glue can be adhered to the microporous array film by gently pressing the attached photoresist microporous array film on the thin layer of adhesive and then peeling off.
  • micro-pit array chip press the micro-hole array with the thin glue adhered on the super-hydrophobic layer, and after standing for more than 4h, remove the micro-pit chip to transfer a super-hydrophobic layer on the upper layer of the micro-hole array.
  • the layer that is, the super-hydrophobic micro-pit array chip.
  • the droplet array can be spontaneously formed.
  • the formation method is as follows: firstly, droplets are dripped by drape, and an aqueous solution is introduced into the micropores by gravity impact force to form an array of droplets in the super-hydrophobic micro-pit array (as shown in FIG. 9); Immersing the entire chip and then withdrawing the excess aqueous solution can spontaneously form an array of droplets in the array of micropits. This process can be repeated.
  • the variance of the droplet volume is reduced from 11% to 1%. This shows that the super-hydrophobic micro-pit array can be quantitatively analyzed. Further, the liquid of the superhydrophobic micro-pit array of the present invention The drop volume is around 50nL, which significantly reduces the consumption of reagents compared to the micro-upgraded volume in conventional multi-well plates, greatly reducing the cost of high-throughput cell assays.
  • DMEM basic (1 ⁇ ) medium (Cat. No. C11995500B7), FBS (Fetal Bovine Serum, fetal calf serum, Cat. No. 10099-141), double antibody (penicillin and streptomycin) (all purchased from Thermal Fisher) .
  • PBS phosphate buffer saline) pH 7.4 basic (1 ⁇ ) (available from Gibco, Life technologies, Cat. No. C10010500BT), Calcein AM (calcium chlorophyll, purchased from Thermal Fisher, Cat. No. C3100MP), PI (bromination) Propionine, purchased from Sigma Aldrich, Cat. No. 25535), Formalin solution (purchased from Sigma Aldrich, Cat. No.
  • Triton-X 100 (purchased from Sigma Aldrich, Cat. No. X100-500ML), BSA (bovine serum albumin, Bovine) Serum Albumin, purchased from AMRESCO, Cat. No.: 0332-100 g), mouse anti-human CD31 mAb (purchased from Sigma, Cat. No. SAB4700463-100 ⁇ g), mouse anti-human VE-cadherin mAb (purchased from Santa Cruz Biotechnology, Cat.
  • the cultivation and detection analysis, the whole cell microarray operation mainly includes super-hydrophobic micro-pits adding liquid, adding cells, forming droplets, dropping culture, changing liquid, and the like, as follows:
  • Droplet formation The droplet array can be spontaneously formed by completely withdrawing the excess medium immersed in the chip.
  • Drop culture In order to inhibit the evaporation of high-throughput cell microarrays in the incubator, a super-hydrophobic chip (shown in Figures 12a, 12b) was placed in a set culture dish and added to the outer culture dish. Sterilize the water to ensure that the air humidity in the microenvironment of the droplet array is saturated.
  • Aspirate the blocking solution dilute the anti-human CD31 antibody 100-fold with blocking solution, add 200 uL to the microwell, and incubate overnight at 4 degrees (add some liquid around, seal with a sealing membrane to prevent evaporation and dry). Wash three times with washing buffer (PBS + 0.05% Tween 20) for 5 minutes each time and shake slowly. Then dilute the secondary antibody 100 times with blocking solution, add 200 uL to the microwell, incubate for 2 hours at room temperature, and take care to avoid light. Wash the wash buffer three times for 5 minutes each time. Add 200 uL of DAPI staining solution to the tissue, incubate for 10 minutes, and wash three times with PBS. Take care to avoid light. Observed by fluorescence microscope. The staining process of VE Cadherin is similar.
  • Vascularization test After the 72h droplet culture is completed, the droplet array is immersed in PBS solution, and the medium in the cleaned micropores is fully diffused and exchanged, then the PBS is removed, and the trypsin solution is added to cover the microwell array. After the cells were digested for 1 min, the trypsin solution was removed, and then the medium was immersed in the medium, and the cells were blown out by pipetting with a pipette, and then the cell solution was transferred to a centrifuge tube, and the excess supernatant was removed after centrifugation. The solution is such that the final cells have an approximate concentration of 1-3 x 10 ⁇ 5 cells. At the same time, the Matrigel-treated culture dish was prepared, and the digested HUVEC cells were added to the Matrigel-treated culture dish, and the culture observation was continued to verify whether the HUVEC cells still have the potential for angiogenesis.
  • hamster kidney cell BHK-21, human umbilical vein epithelial cell HUVEC and human chronic myeloid leukemia cell K562 were cultured in droplets, and the culture conditions were as follows: 37 ° C, 5% CO 2 incubator.
  • the first method of adding cells is to directly immerse the super-hydrophobic micro-pits, and then add 4–6 ⁇ 10 ⁇ 4 cell solutions to the wafer culture dish. After mixing, the sample was allowed to stand for 10 minutes to remove excess cell solution, and a cell solution lattice was formed.
  • the second method of adding cells is to form a droplet array in a super-hydrophobic micro-pit, and then add the cells one by one by a hand-held liquid addition gun.
  • the number of cells required for such a method of addition is very small, and it is expected that the number of cells can be obtained. A small number of precious cell samples play an important role in high-throughput detection.
  • control two ordinary 24-well plate culture
  • Liquid exchange other types of cells except K562 cells need to be changed.
  • the culture fluids of the remaining wells are removed, and the same amount of fresh medium is added to continue at 37 ° C, 5%.
  • the cells were cultured for 12 h in a CO 2 incubator, and then the cell density was counted in 6 wells. The Ln value of the cell relative multiplication ratio was plotted on the ordinate, and the proliferation curve was plotted on the abscissa.
  • the superhydrophobic micro-pit array of the present invention has excellent biocompatibility and can perform high-throughput cell microarray culture for more than 6 days, and can perform high-throughput suspension cell microarray culture which is difficult to realize on most platforms.
  • the superhydrophobic micro-pit array of the present invention is capable of performing high-throughput microarray long-term culture of primary cells and stem cells (shown in Fig.
  • the primary cell microarray on the superhydrophobic micro-pit array of the present invention can also specifically express CD31, VE-cadherin two proteins after 72 hours of droplet culture, and also maintain vascularization.
  • the function (as shown in Figure 14).
  • the superhydrophobic micro-pit array of the invention can realize the physical isolation of the droplet array, so that the red and green fluorescent dye molecules (Rhodamine B and fluorescein isothiocyanate) can be easily added by the spray pattern technology.
  • the spacers are added to different micropits (as shown in Figure 15a) to achieve high throughput detection and analysis of the cellular microarrays.
  • Figure 15a shows that different colors of dye can be added to different columns of micro-pits by spraying, and no cross-contamination occurs between the pits.
  • the present invention can also achieve quantitative addition of a green fluorescent molecule fluorescein isothiocyanate to achieve a compound molecule
  • the concentration gradient was added (shown in Figure 15b and Figure 15c).
  • the super-hydrophobic micro-pit array can automatically form a micro-droplet array in the micro-pit, which is compared with the way of adding cells through a mechanical arm by a mechanical hole in a conventional 384-well plate. More convenient.
  • the droplets on the array of micro-pits are completely physically isolated and can avoid cross-contamination, so it is very suitable for high-throughput screening of soluble factors that are difficult to achieve on traditional high-throughput platforms, such as Screening of high-throughput water-soluble drug molecules.
  • the superhydrophobic micro-pit array of the present invention has excellent biocompatibility, completely avoiding various problems of the prior high-flux cell micro-device: the cell micro-array on the super-hydrophobic micro-pit chip can perform more than six days of droplet type Culture, while the cell culture on the previous micro device can only last for 96 hours, most of which can only be maintained for less than 24 hours; the proliferation rate of cell microarray droplet culture on the super-hydrophobic micro-pit microchip is compared with the ordinary 24-well plate.
  • the proliferation rate of cells cultured on the previous micro-devices is often significantly lower than that of the ordinary well plates; the super-hydrophobic micro-pit array can be used for the construction of primary cells and stem cell microarrays. And culture, and can maintain the original nature of these cells, which is impossible to achieve on most existing micro devices.
  • micro devices may not be able to maintain the original phenotype, making micro devices
  • different functional molecules can be added to different micro-pits by means of spotting. High-throughput cell transfection and combined analysis of stem cell microenvironments have demonstrated that super-hydrophobic micropits are well suited for a variety of high-throughput assays.
  • the greatest advantage of the superhydrophobic micro-pits of the present invention in constructing a cell microarray is that it does not require the expensive robotic device used in the conventional 384-well technique in the process of constructing a cell microarray, so that it can be realized at a very low cost.

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Abstract

La présente invention concerne une puce de réseau de micro-cuvettes super-hydrophobes, son procédé de préparation et ses applications. La puce de réseau de micro-cuvettes comprend une couche de réseau de micro-cuvettes, et la surface de la couche de réseau de micro-cuvettes sur laquelle les micro-cuvettes sont formées, est une surface super-hydrophobe à l'exception des micro-cuvettes ou des fonds des micro-cuvettes. La puce de réseau de micro-cuvettes peut être : 1) la surface à l'exception des micro-cuvettes est la surface super-hydrophobe, et la puce de réseau de micro-cuvettes comprend la couche de réseau de micro-cuvettes et une couche super-hydrophobe fixée à la surface de la couche de réseau de micro-cuvettes ; et 2) la surface à l'exception du fond des micro-cuvettes est une surface super-hydrophobe, la couche de réseau de micro-cuvettes comprend une couche de substrat et une couche de réseau de micropores fixée à la surface d'un substrat, et la couche de réseau de micropores est constituée d'un matériau super-hydrophobe. Au moyen de la puce de réseau de micro-cuvettes, une isolation complète parmi des microréseaux de cellules peut être mise en œuvre à un coût très faible dans le processus de construction de micro-réseaux de cellules, la contamination croisée parmi des réseaux de cellules est évitée, une bonne biocompatibilité est assurée, et la puce de réseau de micro-cuvettes n'a pas d'incidence évidente sur la croissance normale de diverses cellules et est appropriée pour une détection et une analyse de cellules à haut débit.
PCT/CN2017/078946 2016-04-14 2017-03-31 Puce de réseau de micro-cuvettes super-hydrophobes, son procédé de préparation et ses applications WO2017177839A1 (fr)

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