CN110862926A - Multilayer paper chip based on microfluidic technology and construction method thereof - Google Patents
Multilayer paper chip based on microfluidic technology and construction method thereof Download PDFInfo
- Publication number
- CN110862926A CN110862926A CN201810977437.9A CN201810977437A CN110862926A CN 110862926 A CN110862926 A CN 110862926A CN 201810977437 A CN201810977437 A CN 201810977437A CN 110862926 A CN110862926 A CN 110862926A
- Authority
- CN
- China
- Prior art keywords
- paper
- layer
- pdms
- middle layer
- microfluidic
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000005516 engineering process Methods 0.000 title claims abstract description 34
- 238000010276 construction Methods 0.000 title abstract description 15
- 239000007788 liquid Substances 0.000 claims abstract description 105
- 230000002209 hydrophobic effect Effects 0.000 claims abstract description 30
- 238000007789 sealing Methods 0.000 claims abstract description 17
- 239000004205 dimethyl polysiloxane Substances 0.000 claims description 76
- 229920000435 poly(dimethylsiloxane) Polymers 0.000 claims description 76
- 239000003292 glue Substances 0.000 claims description 32
- IJOOHPMOJXWVHK-UHFFFAOYSA-N chlorotrimethylsilane Chemical compound C[Si](C)(C)Cl IJOOHPMOJXWVHK-UHFFFAOYSA-N 0.000 claims description 30
- 239000011521 glass Substances 0.000 claims description 27
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 25
- 239000001301 oxygen Substances 0.000 claims description 25
- 229910052760 oxygen Inorganic materials 0.000 claims description 25
- BGTOWKSIORTVQH-UHFFFAOYSA-N cyclopentanone Chemical compound O=C1CCCC1 BGTOWKSIORTVQH-UHFFFAOYSA-N 0.000 claims description 22
- 238000001035 drying Methods 0.000 claims description 22
- 238000001816 cooling Methods 0.000 claims description 20
- 238000000034 method Methods 0.000 claims description 19
- 239000005051 trimethylchlorosilane Substances 0.000 claims description 15
- 238000001291 vacuum drying Methods 0.000 claims description 10
- 238000010790 dilution Methods 0.000 claims description 8
- 239000012895 dilution Substances 0.000 claims description 8
- 230000004048 modification Effects 0.000 claims description 7
- 238000012986 modification Methods 0.000 claims description 7
- 229920000642 polymer Polymers 0.000 claims description 6
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 claims description 5
- 238000000861 blow drying Methods 0.000 claims description 5
- 229910052802 copper Inorganic materials 0.000 claims description 5
- 239000010949 copper Substances 0.000 claims description 5
- 238000010438 heat treatment Methods 0.000 claims description 5
- 229920002120 photoresistant polymer Polymers 0.000 claims description 5
- 238000000746 purification Methods 0.000 claims description 5
- 230000002441 reversible effect Effects 0.000 claims description 5
- 239000000853 adhesive Substances 0.000 claims description 4
- 230000001070 adhesive effect Effects 0.000 claims description 4
- 239000000463 material Substances 0.000 claims description 4
- 235000013870 dimethyl polysiloxane Nutrition 0.000 claims 12
- CXQXSVUQTKDNFP-UHFFFAOYSA-N octamethyltrisiloxane Chemical compound C[Si](C)(C)O[Si](C)(C)O[Si](C)(C)C CXQXSVUQTKDNFP-UHFFFAOYSA-N 0.000 claims 12
- 238000004987 plasma desorption mass spectroscopy Methods 0.000 claims 12
- 238000001125 extrusion Methods 0.000 claims 1
- 238000003501 co-culture Methods 0.000 abstract description 5
- 238000000059 patterning Methods 0.000 abstract description 4
- 238000004113 cell culture Methods 0.000 abstract description 2
- 238000004519 manufacturing process Methods 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 19
- 238000010586 diagram Methods 0.000 description 9
- 230000002427 irreversible effect Effects 0.000 description 5
- 238000001259 photo etching Methods 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 210000003494 hepatocyte Anatomy 0.000 description 4
- 210000004153 islets of langerhan Anatomy 0.000 description 4
- -1 polydimethylsiloxane Polymers 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 230000009471 action Effects 0.000 description 3
- 239000002390 adhesive tape Substances 0.000 description 3
- 230000004888 barrier function Effects 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 230000010354 integration Effects 0.000 description 3
- 238000000465 moulding Methods 0.000 description 3
- 238000009832 plasma treatment Methods 0.000 description 3
- 239000011148 porous material Substances 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 238000004626 scanning electron microscopy Methods 0.000 description 3
- 238000012512 characterization method Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000004907 flux Effects 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 239000012188 paraffin wax Substances 0.000 description 2
- 238000007639 printing Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- 239000000020 Nitrocellulose Substances 0.000 description 1
- WYTGDNHDOZPMIW-RCBQFDQVSA-N alstonine Natural products C1=CC2=C3C=CC=CC3=NC2=C2N1C[C@H]1[C@H](C)OC=C(C(=O)OC)[C@H]1C2 WYTGDNHDOZPMIW-RCBQFDQVSA-N 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 238000007641 inkjet printing Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 239000002861 polymer material Substances 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 229910052710 silicon Inorganic materials 0.000 description 1
- 239000010703 silicon Substances 0.000 description 1
- 238000004088 simulation Methods 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000009827 uniform distribution Methods 0.000 description 1
Images
Classifications
-
- 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
- C12M23/16—Microfluidic devices; Capillary tubes
-
- 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
- C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Sustainable Development (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Clinical Laboratory Science (AREA)
- Dispersion Chemistry (AREA)
- Immunology (AREA)
- Laminated Bodies (AREA)
Abstract
The invention discloses a multi-layer paper chip based on a microfluidic technology and a construction method thereof, wherein the paper chip is mainly formed by sealing three layers of structures and comprises a lower layer structure, a middle layer structure and an upper layer structure; the lower layer structure is composed of a lower layer liquid inlet (1), a lower layer liquid pool (3) and a lower layer liquid outlet (2), the middle layer structure is composed of two middle layer holes and a middle layer hydrophilic and hydrophobic area (6), the upper layer structure is composed of an upper layer liquid inlet (7), an upper layer liquid pool (9) and an upper layer liquid outlet (8), and the construction method comprises the steps of patterning qualitative filter paper and integrating a microfluidic channel. The invention has simple manufacture, low cost, portability and good reproducibility, and is hopeful to become one of microenvironment equipment for single cell culture and co-culture of various cells.
Description
Technical Field
The invention relates to a photoetching patterning technology and a microfluidic technology, in particular to a multilayer paper chip based on the microfluidic technology and a construction method thereof.
Background
The paper chip uses paper as a substrate material to replace the traditional microfluidic chip using glass, silicon, high polymer materials and the like as substrates, is a device formed by patterning various microfluidic channels on the paper, and is mainly characterized in that liquid is driven to flow through capillary action, and the introduction and reaction of a sample can be completed without external force driving. The paper chip is made of materials which are used up to now, depend on materials with small pore diameter and uniform distribution, such as whatman filter paper, nitrocellulose membrane, cellulose carbonate membrane and the like, and have high cost.
The two-dimensional paper chip manufactured by adopting photoetching technology, paraffin printing, ink-jet printing, drawing, laser processing and other modes is mature, and the patterned paper has higher hydrophilic and hydrophobic resolution ratio and relatively low requirement on equipment by adopting the photoetching technology, so that the low resolution ratio of the paraffin printing and the requirement on the equipment by laser, ink-jet, drawing and the like are avoided. And the two-dimensional paper chip can not realize high-throughput and multi-target detection. The three-dimensional paper chip prepared by the integration method is a novel paper chip developed in recent years, breaks through the limitation, has the characteristics of miniaturization, integration, portability and multifunction, realizes simultaneous analysis of multiple components by integrating a complex network structure, and achieves the purpose of high-throughput and rapid detection.
At present, methods for preparing three-dimensional paper chips include a double-sided adhesive tape multilayer stacking method and a paper folding method, the double-sided adhesive tape multilayer stacking method is single in form, adhesive force of adhesive is weakened due to the fact that paper is bonded through adhesive tapes after long-term storage, the chip structure and subsequent application are affected, the paper folding method needs to consider the problem of mirror images of the paper, design is complex, and operation is inconvenient. The invention adopts common qualitative filter paper and combines the photoetching patterning technology to prepare a uniform, low-cost and high-resolution two-dimensional paper chip, integrates a PDMS polymer with good light transmission, air permeability and biocompatibility on the basis of the two-dimensional paper chip, adopts oxygen and oxygen irreversible sealing treatment to improve the capillary action of a hydrophilic area, and prepares the simple, portable, small sample amount, high flux and good reproducibility three-dimensional paper chip.
Disclosure of Invention
The invention aims to provide a multi-layer paper chip based on a microfluidic technology and a construction method thereof, wherein a PDMS microstructure is integrated, a photoresist soaking technology, a photoetching technology and the microfluidic technology are adopted, the three-dimensional paper chip is simple and portable in integration, small in sample amount, high in flux and good in reproducibility, and the three-dimensional paper chip can be used for 3D culture of cells, cell microenvironment simulation, drug slow release and the like, and has good application value in the fields of medicine and biology.
The invention relates to a multi-layer paper chip based on a microfluidic technology, which is mainly formed by sealing three layers of structures, and is characterized in that: the paper chip is mainly formed by sealing three layers of structures and comprises a lower layer structure, a middle layer structure and an upper layer structure;
the lower layer structure consists of a lower layer liquid inlet, a lower layer liquid pool and a lower layer liquid outlet, wherein the lower layer liquid pool is communicated with the lower layer liquid inlet and the lower layer liquid outlet;
the middle layer structure consists of two middle layer holes and a middle layer hydrophilic and hydrophobic area, and the middle layer hydrophilic and hydrophobic area is not communicated with the middle layer holes;
the upper layer structure consists of an upper layer liquid inlet, an upper layer liquid pool and an upper layer liquid outlet; the upper layer liquid pool is communicated with the upper layer liquid inlet and the upper layer liquid outlet;
the lower layer liquid inlet is communicated with the middle layer hole I, and the lower layer liquid inlet is communicated with the middle layer hole II; the hydrophilic and hydrophobic area of the middle layer, the upper layer liquid pool and the lower layer liquid pool are connected through a channel,
the channel is a straight channel or a curved channel,
the channel is a single channel or a plurality of channels,
the hydrophilic and hydrophobic areas of the middle layer are formed by single circles or formed by a plurality of patterns.
The paper chip is formed by irreversibly sealing an upper layer, a middle layer and a lower layer, wherein the upper layer and the lower layer are made of light-transmitting and air-permeable PDMS polymers, and the middle layer is made of patterned qualitative filter paper.
The intermediate layer filter paper of the paper chip is patterned with an obvious hydrophilic and hydrophobic region through SU8 glue (2025) with a certain dilution multiple, wherein the patterned SU8 glue (2025) is diluted with cyclopentanone, and the volume ratio of the SU8 glue to the cyclopentanone is 1:1-1: 5.
The invention provides a multilayer paper chip based on a microfluidic technology and a construction method thereof, and the basic process is as follows:
(1) patterned qualitative filter paper
Decorating a glass sheet with trimethylchlorosilane steam, drying the glass sheet at 50-95 ℃ for a moment, then throwing a layer of PDMS, heating to solidify the PDMS, placing qualitative filter paper with a certain specification on the PDMS, dripping SU8 glue (2025) with a certain dilution ratio on the paper by using an injector to enable the PDMS to be flat, enabling the filter paper to be soaked by the SU8 glue, defoaming in a vacuum drying oven, drying the paper before 60-95 ℃, extruding the paper by using the glass sheet with the PDMS layer thrown thereon, naturally cooling, exposing, drying after natural cooling, developing, blow-drying, and drying at 50-95 ℃ for 5-30min to obtain the paper with the patterned middle layer for later use;
the trimethyl chlorosilane steam modification time is 1-10min, the thickness of PDMS (polydimethylsiloxane) thrown on a glass sheet is 50-200 mu m, the amount of SU8 glue dripped on qualitative filter paper is 0.6-1.3mL, and the bubble removal temperature range of a vacuum drying oven is 40-70 ℃;
the patterns on the filter paper can be diversified (such as structures formed at circular intervals, straight channel structures, serpentine structures and the like), the developing time is 20-50min, and the oxygen treatment time is 1-3 min;
(2) integrated microfluidic channel
Removing water from the glass sheet for 0.5-2h, performing surface purification treatment on the glass sheet for 1-3min by oxygen, throwing SU8 glue with a certain thickness, pre-baking, performing partial exposure after natural cooling, performing post-baking and development, hardening at 90-180 ℃, naturally cooling to obtain a template of SU8, modifying the template by trimethylchlorosilane for hydrophobization, performing reverse mold treatment on PDMS, curing at 60-95 ℃ to obtain a PDMS chip containing a microfluidic channel structure, performing oxygen treatment on upper and lower PDMS with a structure, performing oxygen treatment on two sides of the paper with the patterned middle layer prepared in the step (1), performing irreversible sealing on the upper and lower PDMS with a channel and the paper with the patterned middle layer, and extruding the integrated paper chip by a copper plate at 60-95 ℃ to smooth and well seal the integrated paper chip, thereby obtaining the three-dimensional paper chip integrated with the microfluidic channel;
the thickness of the SU8 adhesive is 50-500 μm, and PDMS structures containing microfluidic channels can be diversified, such as single straight channels, serpentine channels, parallel multiple channels, and the like;
the time for treating the upper and lower PDMS layers with the structures by oxygen is 0.5-3min, and the time for treating the double-sided oxygen of the paper with the pattern on the middle layer is 1-5 min.
The chip of the invention has simple manufacture, low price, portability and good reproducibility, and is hopeful to become one of microenvironment equipment for single cell culture and co-culture of various cells.
Drawings
Fig. 1 is a schematic view of the overall structure of a three-dimensional paper chip integrated with microfluidic channels according to example 1 of the present invention;
fig. 2a, b and c are schematic structural diagrams of a lower layer, an intermediate layer and an upper layer of a three-dimensional paper chip integrated with a microfluidic channel in case 1 of the present invention, respectively;
FIG. 3 is a scanning electron microscope characterization of the middle layer of an integrated paper chip, demonstrating that the division of the SU8 and paper regions is evident and that SU8 completely saturates the filter paper under experimental conditions;
FIG. 4 is a schematic process diagram of an integrated microfluidic channel;
FIG. 5 is a physical diagram of a novel multi-layer paper chip based on microfluidic technology;
fig. 6 is a partial scanning electron microscopy characterization of the middle layer of the integrated paper chip in example 2.
Fig. 7 is a schematic view of the overall structure of a three-dimensional paper chip integrated with microfluidic channels according to example 3 of the present invention;
fig. 8a, b and c are schematic structural diagrams of a lower layer, an intermediate layer and an upper layer of a three-dimensional paper chip integrated with a microfluidic channel in case 3 of the present invention, respectively;
wherein: 1 is a lower layer liquid inlet, 2 is a lower layer liquid outlet, 3 is a lower layer liquid pool, 4 middle layer holes I, 5 middle layer holes II, 6 middle layer hydrophilic and hydrophobic areas, 7 upper layer liquid inlet, 8 upper layer liquid outlet and 9 upper layer liquid pool.
Detailed Description
The following examples further illustrate the invention but are not intended to limit the invention thereto.
Example 1
Construction of multilayer paper chips and use thereof for isolated culture of hepatocytes
The invention relates to a multi-layer paper chip based on a microfluidic technology and a construction method thereof, the whole is shown in figure 1, and the multi-layer paper chip is formed by sealing three layers of structures, including an upper-layer PDMS structure, a middle paper structure and a lower-layer PDMS structure, and the schematic diagrams are respectively shown in figures 2a, 2b and 2 c. The lower layer structure consists of a lower layer liquid inlet 1, a lower layer liquid pool 3 and a lower layer liquid outlet 2, wherein the lower layer liquid pool 3 is communicated with the lower layer liquid inlet 1 and the lower layer liquid outlet 2;
the middle layer structure is composed of a middle layer hole I4, a middle layer hole II 5 and a middle layer hydrophilic and hydrophobic area 6, and the middle layer hydrophilic and hydrophobic area 6 is not communicated with the middle layer hole;
the upper layer structure consists of an upper layer liquid inlet 7, an upper layer liquid pool 9 and an upper layer liquid outlet 8; the upper layer liquid pool (9) is communicated with the upper layer liquid inlet (7) and the upper layer liquid outlet (8);
the lower layer liquid inlet 1 is communicated with the middle layer hole 4, and the lower layer liquid inlet 2 is communicated with the middle layer hole 5; the middle layer hydrophilic and hydrophobic area 6, the upper layer liquid pool 3 and the lower layer liquid pool 9 are connected through a channel,
the channel is a straight channel, the channel is a single channel,
the middle layer hydrophilic and hydrophobic region 6 is formed by a single circle.
The multi-layer paper chip based on the microfluidic technology is formed by irreversibly sealing an upper layer, a middle layer and a lower layer, wherein the upper layer and the lower layer are made of light-transmitting and air-permeable PDMS polymers, and the middle layer is made of photoresist patterned qualitative filter paper.
SU8 glue (2025) patterned by filter paper at the middle layer of the paper chip is diluted by cyclopentanone, and the volume ratio of the SU8 glue to the cyclopentanone is 1: 1.
The paper chip has an obvious hydrophilic and hydrophobic area after the middle layer filter paper is patterned, and the upper layer, the lower layer and the two surfaces of the middle layer paper are sealed after plasma treatment for 1.5 min.
The invention provides a multilayer paper chip construction method based on a microfluidic technology, which comprises the following basic processes: (1) patterned qualitative filter paper
Decorating a glass sheet with trimethylchlorosilane steam, drying the glass sheet at 65 ℃ for a moment, then throwing a layer of PDMS, heating to solidify the PDMS, placing qualitative filter paper with a certain specification on the PDMS, dripping SU8 glue (2025) with a certain dilution ratio (1:1) on the paper by using an injector to enable the paper to be flat and enable the filter paper to be soaked by the SU8 glue, removing bubbles in a vacuum drying oven, drying the paper at 65 ℃ in advance, extruding the paper with the PDMS layer, naturally cooling, exposing, drying after, developing after natural cooling, blow-drying, and drying at 65 ℃ for 5min to obtain paper with a patterned middle layer for later use;
the trimethyl chlorosilane steam modification time is 5min, the thickness of PDMS (polydimethylsiloxane) thrown on a glass sheet is 100 mu m, the amount of SU8 glue dripped on qualitative filter paper is 0.8mL, and the bubble removal temperature range of a vacuum drying oven is 45 ℃;
the pattern on the filter paper is as shown in figure 2b, the structure is formed at circular intervals, the aperture of the hydrophilic area is 400 mu m, the pore interval is 200 mu m, and the developing time is 30 min; the structure of the middle layer paper prepared by the above method was characterized by scanning electron microscopy, and the results are shown in fig. 3.
(2) Integrated microfluidic channel
Removing water from the glass sheet for 0.5h, performing surface purification treatment on the glass sheet for 1.5min by oxygen, throwing SU8 glue with a certain thickness, pre-drying, performing partial exposure after natural cooling, performing post-drying, developing, hardening at 160 ℃, obtaining a template of SU8 after natural cooling, modifying the template by trimethylchlorosilane for hydrophobization, performing reverse molding by PDMS, curing at 65 ℃ to obtain a PDMS chip containing a microfluidic channel structure, performing oxygen treatment on upper and lower PDMS layers with a structure, performing oxygen treatment on the two surfaces of the paper with the patterned middle layer prepared in the step (1), performing irreversible sealing on the upper and lower PDMS with a channel and the paper with the patterned middle layer, and extruding the integrated paper chip by a copper plate at 80 ℃ to make the integrated paper chip flat and well sealed, thus obtaining the paper chip with the integrated microfluidic channel, wherein the preparation process is shown in FIG. 4;
the thickness of the SU8 gel is 150 μm, the PDMS structure containing the microfluidic channel is a single straight channel, as shown in FIGS. 2a and 2c, the channels of the PDMS microfluidic liquid pool (3) and (9) are 10mm long and 3mm wide;
the time for treating the upper and lower PDMS layers with the structures by oxygen is 0.5min, and the time for treating the double-sided oxygen of the paper with the pattern on the middle layer is 1.5 min. The schematic diagram of the multi-layer paper chip based on the microfluidic technology prepared by the above steps is shown in fig. 5.
Compared with the traditional two-dimensional paper chip, the novel multilayer paper chip based on the microfluidic technology has the following advantages: the co-culture of 2 cells can be realized, different cells are inoculated in the upper layer liquid pool and the lower layer liquid pool, and the interaction between the cells is researched; the paper chip of the integrated microfluidic channel is a perfusable system, namely liquid can be perfused from a lower layer liquid inlet and an upper layer liquid inlet, so that a microenvironment is simulated, and more favorable nutrient substances for cell growth are provided; the construction of the three-dimensional effect is a technical platform for constructing barrier research, namely, cells can be loaded on two sides of a middle paper chip to form barriers, and nanoparticles and medicines can be added from an inlet of a lower layer or an upper layer to further research the action mechanism of the barriers and the like.
The prepared chip is used for independent culture of the hepatic cells. But also can be used for culturing other kinds of cells independently, and the invention is not limited. Here, HepG2 was selected as the hepatocyte, and HepG2 cells (cell density 4X 10) were seeded at the upper liquid inlet (7)5) The cells are cultured individually, and will not be described in detail here.
Example 2
Constructing multilayer paper chip and using for co-culture of liver and pancreatic island cells
The invention relates to a method for constructing a multilayer paper chip based on a microfluidic technology, which is integrally shown in figure 1 and formed by sealing three layers of structures, wherein the structures comprise an upper-layer PDMS structure, a middle-layer paper structure and a lower-layer PDMS structure, and the schematic diagrams are respectively shown in figures 2a, 2b and 2 c. The lower layer structure consists of a lower layer liquid inlet 1, a lower layer liquid pool 3 and a lower layer liquid outlet 2, wherein the lower layer liquid pool 3 is communicated with the lower layer liquid inlet 1 and the lower layer liquid outlet 2;
the middle layer structure consists of two middle layer holes 4 and 5 and a middle layer hydrophilic and hydrophobic area 6, and the middle layer hydrophilic and hydrophobic area 6 is not communicated with the middle layer holes 4 and 5;
the upper layer structure consists of an upper layer liquid inlet 7, an upper layer liquid pool 9 and an upper layer liquid outlet 8; the upper layer liquid pool 9 is communicated with the upper layer liquid inlet 7 and the upper layer liquid outlet 8;
the lower layer liquid inlet 1 is communicated with the middle layer hole 4, and the lower layer liquid inlet 2 is communicated with the middle layer hole 5; the middle layer hydrophilic and hydrophobic area 6, the upper layer liquid pool 3 and the lower layer liquid pool 9 are connected through a channel,
the channel is a straight channel, the channel is a single channel,
the middle layer hydrophilic and hydrophobic region 6 is formed by a single circle.
The multi-layer paper chip based on the microfluidic technology is formed by irreversibly sealing an upper layer, a middle layer and a lower layer, wherein the upper layer and the lower layer are made of light-transmitting and air-permeable PDMS polymers, and the middle layer is made of photoresist patterned qualitative filter paper.
SU8 glue (2025) patterned by filter paper at the middle layer of the paper chip is diluted by cyclopentanone, and the volume ratio of the SU8 glue to the cyclopentanone is 1: 3.
The paper chip has an obvious hydrophilic and hydrophobic area after the middle layer filter paper is patterned, and the upper layer, the lower layer and the two surfaces of the middle layer paper are sealed after plasma treatment for 1.0 min.
The invention provides a multilayer paper chip based on a microfluidic technology and a construction method thereof, and the basic process is as follows:
(1) patterned qualitative filter paper
Modifying a glass sheet by using trimethylchlorosilane steam, drying the glass sheet at 95 ℃ for a moment, then throwing a layer of PDMS, heating to solidify the PDMS, placing qualitative filter paper with a certain specification on the PDMS, dripping SU8 glue (2025) with a certain dilution ratio (1:3) on the paper by using an injector to enable the paper to be flat and enable the filter paper to be soaked by the SU8 glue, removing bubbles in a vacuum drying oven, drying the paper at 95 ℃ in advance, extruding the paper by using the glass sheet with the PDMS layer thrown thereon, naturally cooling, exposing, drying after naturally cooling, developing, blow-drying, and drying at 95 ℃ for 10min to obtain paper with a patterned middle layer for later use;
the trimethyl chlorosilane steam modification time is 5min, the thickness of PDMS (polydimethylsiloxane) thrown on a glass sheet is 80 mu m, the amount of SU8 glue dripped on qualitative filter paper is 0.8mL, and the bubble removal temperature range of a vacuum drying oven is 45 ℃;
the pattern on the filter paper is as shown in figure 2b, the structure is formed at circular intervals, the aperture of the hydrophilic area is 400 mu m, the pore interval is 200 mu m, and the developing time is 30 min; the structure of the middle layer paper prepared by the above method was characterized by scanning electron microscopy, and the results are shown in fig. 6. The SU8 patterned paper diluted by cyclopentanone has clearer outline and better effect.
(2) Integrated microfluidic channel
Removing water from a glass sheet for 2 hours, performing surface purification treatment by oxygen for 1min, throwing SU8 glue with a certain thickness, prebaking, naturally cooling, then performing partial exposure, prebaking, developing, hardening at 120 ℃, naturally cooling to obtain a SU8 template, performing modification by trimethylchlorosilane to make the template hydrophobic, performing reverse molding by PDMS, curing at 95 ℃ to obtain a PDMS chip containing a microfluidic channel structure, performing oxygen treatment on upper and lower PDMS layers with structures, performing oxygen treatment on the two surfaces of the paper with the patterned middle layer prepared in the step (1), performing irreversible sealing on the upper and lower PDMS layers with channels and the paper with the patterned middle layer, and extruding by a copper plate at 95 ℃ to make the integrated paper chip flat and well sealed, thus obtaining the multi-layer paper chip integrating the microfluidic channel, wherein the preparation process is shown in figure 4;
the thickness of the SU8 gel is 200 μm, the PDMS structure containing the microfluidic channel is a single straight channel, as shown in FIGS. 2a and 2c, the channels of the PDMS microfluidic liquid pool (3) and (9) are 10mm long and 3 μm wide;
the time for treating the upper and lower PDMS layers with the structures by oxygen is 0.5min, and the time for treating the double-sided oxygen of the paper with the pattern on the middle layer is 1.0 min. The schematic diagram of the multi-layer paper chip based on the microfluidic technology prepared by the above steps is shown in fig. 5.
The invention is not limited in that HepG2 is selected as the hepatic cell, β -TC6 is selected as the islet cell, β -TC6 cells (cell density is 1 multiplied by 10) are inoculated at the inlet 1 of the lower layer liquid6) HepG2 cells (cell density 1X 10) were seeded at the upper liquid inlet 76) The co-culture of the cells is carried out and will not be described in detail here.
Example 3
Construction of a multilayer paper chip and use in islet cell isolation
The invention relates to a multi-layer paper chip based on a microfluidic technology and a construction method thereof, the whole is shown in figure 7, and the multi-layer paper chip is formed by sealing three layers of structures, including an upper-layer PDMS structure, a middle paper structure and a lower-layer PDMS structure, and the schematic diagrams are respectively shown in figures 8a, 8b and 8 c. The lower layer structure consists of a lower layer liquid inlet 1, a lower layer liquid pool 3 and a lower layer liquid outlet 2, wherein the lower layer liquid pool 3 is communicated with the lower layer liquid inlet 1 and the lower layer liquid outlet 2;
the middle layer structure is composed of a middle layer hole I4, a middle layer hole II 5 and a middle layer hydrophilic and hydrophobic area 6, and the middle layer hydrophilic and hydrophobic area 6 is not communicated with the middle layer hole;
the upper layer structure consists of an upper layer liquid inlet 7, an upper layer liquid pool 9 and an upper layer liquid outlet 8; the upper layer liquid pool 9 is communicated with the upper layer liquid inlet 7 and the upper layer liquid outlet 8;
the lower layer liquid inlet 1 is communicated with the middle layer hole 4, and the lower layer liquid inlet 2 is communicated with the middle layer hole 5; the middle layer hydrophilic and hydrophobic area 6, the upper layer liquid pool 3 and the lower layer liquid pool 9 are connected through a channel,
the channel is a through channel, the channel is a single channel,
the hydrophilic and hydrophobic region 6 of the middle layer is composed of array straight channels.
The multi-layer paper chip based on the microfluidic technology is formed by irreversibly sealing an upper layer, a middle layer and a lower layer, wherein the upper layer and the lower layer are made of light-transmitting and air-permeable PDMS polymers, and the middle layer is made of photoresist patterned qualitative filter paper.
SU8 glue (2025) patterned by filter paper at the middle layer of the paper chip is diluted by cyclopentanone, and the volume ratio of the SU8 glue to the cyclopentanone is 1: 5.
The paper chip has an obvious hydrophilic and hydrophobic area after the middle layer filter paper is patterned, and the upper layer, the lower layer and the two surfaces of the middle layer paper are sealed after being subjected to plasma treatment for 2.0 min.
The invention provides a multilayer paper chip construction method based on a microfluidic technology, which comprises the following basic processes:
(1) patterned qualitative filter paper
Modifying a glass sheet by using trimethylchlorosilane steam, drying the glass sheet at 80 ℃ for a moment, then throwing a layer of PDMS, heating to solidify the PDMS, placing qualitative filter paper with a certain specification on the PDMS, dripping SU8 glue (2025) with a certain dilution ratio (1:5) on the paper by using an injector to enable the paper to be flat and enable the filter paper to be soaked by the SU8 glue, removing bubbles in a vacuum drying oven, drying the paper at 80 ℃, extruding the paper by using the glass sheet with the PDMS layer, naturally cooling, exposing, drying, developing after natural cooling, blow-drying, and drying for 15min at 80 ℃ to obtain paper with a patterned middle layer for later use;
the trimethyl chlorosilane steam modification time is 5min, the thickness of PDMS (polydimethylsiloxane) thrown on a glass sheet is 200 mu m, the amount of SU8 glue dripped on qualitative filter paper is 1.3mL, and the bubble removal temperature range of a vacuum drying oven is 65 ℃;
the pattern on the filter paper is as shown in FIG. 8b, and the structure formed by the array of straight channels, the length of the straight channel is 6mm, the width is 300 μm, the interval is 300 μm, and the developing time is 20 min.
(2) Integrated microfluidic channel
Removing water from the glass sheet for 1.5h, performing surface purification treatment for 2min by oxygen, throwing SU8 glue with a certain thickness, prebaking, naturally cooling, then performing partial exposure, postbaking, developing, hardening at 180 ℃, naturally cooling to obtain a SU8 template, performing modification by trimethylchlorosilane for hydrophobization, performing reverse molding by PDMS, curing at 80 ℃ to obtain a PDMS chip containing a microfluidic channel structure, performing oxygen treatment on upper and lower PDMS layers with structures, performing oxygen treatment on the two surfaces of the paper with the patterned middle layer prepared in the step (1), performing irreversible sealing on the upper and lower PDMS layers with channels and the paper with the patterned middle layer, and extruding by a copper plate at 80 ℃ to ensure that the integrated paper chip is flat and well sealed, thus obtaining the multi-layer paper chip with integrated microfluidic channels, wherein the preparation process is as shown in figure 4;
the thickness of the SU8 gel is 500 μm, the PDMS structure containing the microfluidic channel is a single straight channel, as shown in FIGS. 2a and 2c, the channels of the PDMS microfluidic liquid pool (3) and (9) are 10mm long and 3mm wide;
the time for treating the upper and lower PDMS layers with the structures by oxygen is 0.5min, and the time for treating the double-sided oxygen of the paper with the pattern on the middle layer is 2.0 min.
Islet cells were cultured individually using the prepared chip, and islet cells (cell density 2X 10) were seeded at the upper liquid inlet (7)7) The cells are cultured individually, and will not be described in detail here.
Claims (9)
1. A multi-layer paper chip based on a microfluidic technology is characterized in that: the paper chip is mainly formed by sealing three layers of structures and comprises a lower layer structure, a middle layer structure and an upper layer structure;
the lower layer structure consists of a lower layer liquid inlet (1), a lower layer liquid pool (3) and a lower layer liquid outlet (2), wherein the lower layer liquid pool (3) is communicated with the lower layer liquid inlet (1) and the lower layer liquid outlet (2);
the middle layer structure consists of a middle layer hole I (4), a middle layer hole II (5) and a middle layer hydrophilic and hydrophobic area (6), and the middle layer hydrophilic and hydrophobic area (6) is not communicated with the middle layer hole;
the upper layer structure consists of an upper layer liquid inlet (7), an upper layer liquid pool (9) and an upper layer liquid outlet (8); the upper layer liquid pool (9) is communicated with the upper layer liquid inlet (7) and the upper layer liquid outlet (8);
the lower layer liquid inlet (1) is communicated with the middle layer hole (4), and the lower layer liquid inlet (2) is communicated with the middle layer hole (5); the middle hydrophilic and hydrophobic region (6), the upper liquid pool (3) and the lower liquid pool (9) are connected through a channel,
the channel is a straight channel or a curved channel,
the channel is a single channel or a plurality of channels,
the hydrophilic and hydrophobic areas (6) of the middle layer are formed by single circles or various patterns.
2. A microfluidic-based multilayer paper chip according to claim 1, wherein: the paper chip is formed by irreversibly sealing an upper layer, a middle layer and a lower layer, wherein the upper layer and the lower layer are made of light-transmitting and air-permeable PDMS polymers, and the middle layer is made of photoresist patterned qualitative filter paper.
3. A microfluidic-based multilayer paper chip according to claim 2, wherein: the intermediate layer material of the paper chip, qualitative filter paper, is patterned with obvious hydrophilic and hydrophobic areas through SU8 glue with a certain dilution factor.
4. A microfluidic-based multilayer paper chip according to claim 3, wherein: the patterned SU8 glue was diluted with cyclopentanone, and the volume ratio of SU8 glue to cyclopentanone was 1: 1-5.
5. The method for constructing the multilayer paper chip based on the microfluidic technology as claimed in claim 1, is characterized by comprising the following steps:
(1) patterned qualitative filter paper
Modifying a glass sheet by using trimethylchlorosilane steam, drying the glass sheet at 50-95 ℃ for a moment, then throwing a layer of PDMS, heating to solidify the PDMS, placing qualitative filter paper with a certain specification on the PDMS, dripping SU8 glue with a certain dilution multiple on the paper by using an injector to enable the paper to be flat and enable the filter paper to be soaked by the SU8 glue, defoaming in a vacuum drying oven, pre-drying at 60-95 ℃, extruding the paper by using the glass sheet with the PDMS layer, naturally cooling, exposing, post-drying, naturally cooling, developing, blow-drying, and drying at 60-95 ℃ for 5-30min to obtain paper with a patterned middle layer for later use;
(2) integrated microfluidic channel
The glass sheet is dewatered for 0.5-2h, surface purification treatment is carried out for 1-3min by oxygen, SU8 glue with certain thickness is thrown, prebaking is carried out, partial exposure is carried out after natural cooling, postbaking and developing are carried out, hardening is carried out at 90-180 ℃, a template of SU8 is obtained after natural cooling, in order to make the template hydrophobic, trimethylchlorosilane is used for modifying, PDMS reverse mould is used, curing is carried out at 60-95 ℃ to obtain the PDMS chip containing the microfluidic channel structure, the upper and lower PDMS with the structure and the double surfaces of the middle layer patterned paper prepared in the step (1) are treated by oxygen, then the upper and lower PDMS with the channel and the middle layer patterned paper are irreversibly sealed, the copper plate is used for extrusion at 60-95 ℃ to ensure that the integrated paper chip is flat and well sealed, and the multilayer paper chip integrating the microfluidic channel is obtained.
6. The method for constructing a multi-layer paper chip based on the microfluidic technology as claimed in claim 5, wherein in the step (1): the modification time of the trimethylchlorosilane steam is 1-10min, the thickness of the PDMS spun on the glass sheet is 50-200 mu m, the amount of SU8 glue with certain dilution times dripped on qualitative filter paper is 0.6-1.3mL, and the bubble removing temperature range of the vacuum drying oven is 40-70 ℃.
7. The method for constructing a multi-layer paper chip based on the microfluidic technology as claimed in claim 5, wherein in the step (1): the pattern on the paper patterned by the middle layer is a structure formed by circular intervals, a straight channel structure, a snake-shaped structure or other structures, and the developing time is 20-50 min.
8. The method for constructing a multi-layer paper chip based on the microfluidic technology as claimed in claim 5, wherein in the step (2): the thickness of the SU8 adhesive is 50-500 μm, and the channels of the PDMS chip containing the microfluidic channels are single straight channels, serpentine channels, parallel channels or channels with other structures.
9. The method for constructing the multilayer paper chip based on the microfluidic technology as claimed in claim 5, wherein in the step (2): the time for oxygen-treating the PDMS on the upper layer and the lower layer with the structures is 0.5-3min, and the time for oxygen-treating the double surfaces of the paper with the pattern on the middle layer is 1-5 min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810977437.9A CN110862926A (en) | 2018-08-27 | 2018-08-27 | Multilayer paper chip based on microfluidic technology and construction method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810977437.9A CN110862926A (en) | 2018-08-27 | 2018-08-27 | Multilayer paper chip based on microfluidic technology and construction method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110862926A true CN110862926A (en) | 2020-03-06 |
Family
ID=69650852
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810977437.9A Pending CN110862926A (en) | 2018-08-27 | 2018-08-27 | Multilayer paper chip based on microfluidic technology and construction method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110862926A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113304789A (en) * | 2021-05-21 | 2021-08-27 | 合肥工业大学 | Manufacturing method of pump-free composite microfluidic chip with SERS substrate |
| CN114196504A (en) * | 2020-09-17 | 2022-03-18 | 中国科学院大连化学物理研究所 | Exosome enrichment chip based on chitosan positive and negative charge adsorption principle and preparation method thereof |
| CN118322551A (en) * | 2024-04-25 | 2024-07-12 | 南京航空航天大学 | Rapid preparation method of multilayer microfluidic chip based on photo-curing molding technology |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110105360A1 (en) * | 2008-03-27 | 2011-05-05 | President And Fellows Of Harvard College | Paper-based cellular arrays |
| CN105420106A (en) * | 2015-11-23 | 2016-03-23 | 大连医科大学附属第一医院 | Bionic glomerulus chip and chipset composed of bionic glomerulus chips |
| CN106811413A (en) * | 2015-11-30 | 2017-06-09 | 中国科学院大连化学物理研究所 | Multiple organ chip based on microflow control technique and preparation method thereof |
| US20180017585A1 (en) * | 2016-07-12 | 2018-01-18 | Emulate Inc. | Additive channels |
| CN107904172A (en) * | 2017-12-26 | 2018-04-13 | 大连理工大学 | A kind of integrated form cell three-dimensional dynamic cultivation micro element and preparation method thereof |
| CN107937242A (en) * | 2017-10-17 | 2018-04-20 | 浙江大学 | A kind of DNA electricity solid phase extraction method and device based on paper substrate |
-
2018
- 2018-08-27 CN CN201810977437.9A patent/CN110862926A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110105360A1 (en) * | 2008-03-27 | 2011-05-05 | President And Fellows Of Harvard College | Paper-based cellular arrays |
| CN105420106A (en) * | 2015-11-23 | 2016-03-23 | 大连医科大学附属第一医院 | Bionic glomerulus chip and chipset composed of bionic glomerulus chips |
| CN106811413A (en) * | 2015-11-30 | 2017-06-09 | 中国科学院大连化学物理研究所 | Multiple organ chip based on microflow control technique and preparation method thereof |
| US20180017585A1 (en) * | 2016-07-12 | 2018-01-18 | Emulate Inc. | Additive channels |
| CN107937242A (en) * | 2017-10-17 | 2018-04-20 | 浙江大学 | A kind of DNA electricity solid phase extraction method and device based on paper substrate |
| CN107904172A (en) * | 2017-12-26 | 2018-04-13 | 大连理工大学 | A kind of integrated form cell three-dimensional dynamic cultivation micro element and preparation method thereof |
Non-Patent Citations (1)
| Title |
|---|
| RAHIM RAHIMI等: ""Paper-based in-vitro model for on-chip investigation of the human respiratory system"", 《LAB ON A CHIP》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114196504A (en) * | 2020-09-17 | 2022-03-18 | 中国科学院大连化学物理研究所 | Exosome enrichment chip based on chitosan positive and negative charge adsorption principle and preparation method thereof |
| CN113304789A (en) * | 2021-05-21 | 2021-08-27 | 合肥工业大学 | Manufacturing method of pump-free composite microfluidic chip with SERS substrate |
| CN118322551A (en) * | 2024-04-25 | 2024-07-12 | 南京航空航天大学 | Rapid preparation method of multilayer microfluidic chip based on photo-curing molding technology |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101629143B (en) | Microfluidic cell array chip for high-throughput drug screening, method and application | |
| Khoury et al. | Ultra rapid prototyping of microfluidic systems using liquid phase photopolymerization | |
| US20140273223A1 (en) | Micro-device for culturing cells, method for manufacturing same, and method for culturing cells using the micro-device for culturing cells | |
| DE112011104891T5 (en) | 3D microfluidic devices based on openwork thermoplastic elastomer membranes | |
| CN103421691B (en) | Glass chip for cultivating single cell array based on microfluidic patterning technology and preparation method thereof | |
| Selimović et al. | Microfabricated polyester conical microwells for cell culture applications | |
| CN109234163B (en) | High-throughput tumor targeted drug concentration screening microfluidic device | |
| CN110862926A (en) | Multilayer paper chip based on microfluidic technology and construction method thereof | |
| CN111438859A (en) | Patterned nano array template and preparation method and application thereof | |
| KR101985311B1 (en) | 3 Dimensional Cell Culture Microchip Containing Nano-porous Membrane And Manufacturing Method Thereof | |
| JP5078073B2 (en) | Method for producing a resin film having a three-dimensional structure | |
| Kobayashi et al. | Preparation of microfluidic devices using micropatterning of a photosensitive material by a maskless, liquid‐crystal‐display projection method | |
| KR20120011609A (en) | Microfluidic platform and preparation method of the same | |
| Farshidfar et al. | The feasible application of microfluidic tissue/organ-on-a-chip as an impersonator of oral tissues and organs: a direction for future research | |
| KR102037595B1 (en) | Hydrogel membrane fixed vertically in microfluidic chip and preparation method thereof | |
| CN109797096A (en) | Digital pcr chip and preparation method thereof, preparation facilities, application method | |
| CN112920951B (en) | Cell screening chip and manufacturing and cell screening and collecting method thereof | |
| CN107824105B (en) | Micro mixer capable of adjusting solution concentration gradient based on moving bubbles | |
| CN202433389U (en) | Microfluidic chip with micro-channel with high depth-to-width ratio | |
| CN108312659A (en) | Pattern array of bubbles and its preparation method and application | |
| CN108545692A (en) | A kind of facture of microchip method of vias inner walls coating Parylene | |
| DE102015015535A1 (en) | Microstructured polymer body, microbioreactor and process for its preparation | |
| CN112051631B (en) | Preparation method of micro-lens array film | |
| de Haan et al. | Facile fabrication of microperforated membranes with re-useable SU-8 molds for organs-on-chips | |
| KR102146284B1 (en) | Apparatus of forming liquid-mediated material pattern, method of manufacturing the same, method of forming liquid-mediated pattern using the same, and liquid-mediated pattern |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20200306 |
|
| RJ01 | Rejection of invention patent application after publication |