CN109797096A - Digital pcr chip and preparation method thereof, preparation facilities, application method - Google Patents
Digital pcr chip and preparation method thereof, preparation facilities, application method Download PDFInfo
- Publication number
- CN109797096A CN109797096A CN201910070932.6A CN201910070932A CN109797096A CN 109797096 A CN109797096 A CN 109797096A CN 201910070932 A CN201910070932 A CN 201910070932A CN 109797096 A CN109797096 A CN 109797096A
- Authority
- CN
- China
- Prior art keywords
- sample
- digital pcr
- substrate
- pcr chip
- imprinting
- 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.)
- Granted
Links
- 238000000034 method Methods 0.000 title claims abstract description 40
- 238000002360 preparation method Methods 0.000 title abstract description 19
- 239000000758 substrate Substances 0.000 claims abstract description 59
- 238000006243 chemical reaction Methods 0.000 claims abstract description 30
- 238000004519 manufacturing process Methods 0.000 claims abstract description 12
- 238000007847 digital PCR Methods 0.000 claims description 93
- 239000003292 glue Substances 0.000 claims description 42
- 229920002799 BoPET Polymers 0.000 claims description 29
- 238000000465 moulding Methods 0.000 claims description 25
- 238000007789 sealing Methods 0.000 claims description 22
- 239000011248 coating agent Substances 0.000 claims description 18
- 238000000576 coating method Methods 0.000 claims description 18
- 238000004804 winding Methods 0.000 claims description 17
- 239000000853 adhesive Substances 0.000 claims description 15
- 230000001070 adhesive effect Effects 0.000 claims description 15
- 238000003825 pressing Methods 0.000 claims description 11
- 238000012546 transfer Methods 0.000 claims description 8
- 238000010438 heat treatment Methods 0.000 claims description 7
- 239000002480 mineral oil Substances 0.000 claims description 6
- 235000010446 mineral oil Nutrition 0.000 claims description 6
- 238000005096 rolling process Methods 0.000 claims description 6
- 229920005570 flexible polymer Polymers 0.000 claims description 5
- 230000009471 action Effects 0.000 claims description 4
- 230000001678 irradiating effect Effects 0.000 claims description 2
- 150000001875 compounds Chemical class 0.000 claims 1
- 238000001514 detection method Methods 0.000 abstract description 21
- 239000010408 film Substances 0.000 description 29
- 238000003752 polymerase chain reaction Methods 0.000 description 21
- 239000002184 metal Substances 0.000 description 20
- 230000008569 process Effects 0.000 description 15
- 238000005516 engineering process Methods 0.000 description 14
- 230000004907 flux Effects 0.000 description 8
- 230000005540 biological transmission Effects 0.000 description 6
- 238000010586 diagram Methods 0.000 description 5
- 230000003321 amplification Effects 0.000 description 4
- 238000010923 batch production Methods 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 238000003199 nucleic acid amplification method Methods 0.000 description 4
- 229920000139 polyethylene terephthalate Polymers 0.000 description 4
- 239000005020 polyethylene terephthalate Substances 0.000 description 4
- 239000011148 porous material Substances 0.000 description 4
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 150000007523 nucleic acids Chemical class 0.000 description 3
- 102000039446 nucleic acids Human genes 0.000 description 3
- 108020004707 nucleic acids Proteins 0.000 description 3
- 229920000671 polyethylene glycol diacrylate Polymers 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 229910052710 silicon Inorganic materials 0.000 description 3
- 239000010703 silicon Substances 0.000 description 3
- XMLYCEVDHLAQEL-UHFFFAOYSA-N 2-hydroxy-2-methyl-1-phenylpropan-1-one Chemical compound CC(C)(O)C(=O)C1=CC=CC=C1 XMLYCEVDHLAQEL-UHFFFAOYSA-N 0.000 description 2
- 239000002202 Polyethylene glycol Substances 0.000 description 2
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000012864 cross contamination Methods 0.000 description 2
- 125000004386 diacrylate group Chemical group 0.000 description 2
- 238000001125 extrusion Methods 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000001127 nanoimprint lithography Methods 0.000 description 2
- 231100000252 nontoxic Toxicity 0.000 description 2
- 230000003000 nontoxic effect Effects 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 239000003504 photosensitizing agent Substances 0.000 description 2
- 229920003229 poly(methyl methacrylate) Polymers 0.000 description 2
- 229920001223 polyethylene glycol Polymers 0.000 description 2
- -1 polyethylene terephthalate Polymers 0.000 description 2
- 239000004926 polymethyl methacrylate Substances 0.000 description 2
- 238000007493 shaping process Methods 0.000 description 2
- 230000003068 static effect Effects 0.000 description 2
- 230000009967 tasteless effect Effects 0.000 description 2
- 239000010409 thin film Substances 0.000 description 2
- 238000002834 transmittance Methods 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 1
- 239000002390 adhesive tape Substances 0.000 description 1
- 238000003491 array Methods 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000001312 dry etching Methods 0.000 description 1
- 230000005611 electricity Effects 0.000 description 1
- 238000005530 etching Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 238000001917 fluorescence detection Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 238000001259 photo etching Methods 0.000 description 1
- 238000009832 plasma treatment Methods 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
- 239000002210 silicon-based material Substances 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 238000000233 ultraviolet lithography Methods 0.000 description 1
Landscapes
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Shaping Of Tube Ends By Bending Or Straightening (AREA)
Abstract
The present invention provides a kind of digital pcr chips and preparation method thereof, preparation facilities, application method, digital pcr chip includes: the structure sheaf (12) of substrate (11) and setting above substrate (11), structure sheaf (12) has at least one sample channel, each sample channel includes the microcellular structure with multiple micropores (121) and the spill cavity (122) for holding extra sample, and spill cavity (122) is located on at least side of microcellular structure.The case where technical solution of the present invention prevents extra sample from overflowing, it is easy to use, detection efficiency is improved, low manufacture cost, yield are high, improve sample throughput, improve PCR reaction heat conduction efficiency.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a digital PCR chip and a preparation method, a preparation device and a use method thereof.
Background
Polymerase Chain Reaction (PCR), which is a nucleic acid detection technology, has been the most important support technology in the fields of life science research and clinical molecular diagnosis since birth, and has greatly promoted the development of various fields of life science. Over the short thirty years, PCR technology has evolved from the first to the third generation, referred to as Digital PCR (dPCR), by distributing the sample into a large number of reaction units, each containing one or more copies of a target molecule (DNA template), performing PCR amplification on the target molecule, and performing statistical analysis on the fluorescent signal of the reaction units after the amplification is complete. The digital PCR technology does not need a standard curve and a reference, is insensitive to an inhibitor influencing the PCR reaction efficiency, and is an absolute quantitative technology of nucleic acid molecules. Compared with the traditional PCR technology, the digital PCR technology has the advantages of high sensitivity, high specificity, accurate quantification and the like, and is widely applied to the aspects of trace nucleic acid sample detection, rare mutation detection under a complex background, small difference identification of expression quantity, copy number variation detection and the like.
At present, digital PCR technology platforms mainly have three types: a large-scale integrated micro-fluidic chip, a liquid drop and a micro-cavity type digital PCR system. The micro-cavity type digital PCR is based on a micro-nano processing technology, a high-density micro-reaction cavity is processed on a chip in advance, and the micro-reaction cavity is used for dividing a sample to form reaction units which are mutually independent in space, so that the sample can be kept completely isolated, cross contamination can be effectively prevented, and pipetting operation can be reduced. The volume and the position of the micro-reaction cavity are fixed, so that real-time fluorescence detection is convenient to realize, the requirement on a detection system is reduced, and the precision and the stability of detection are improved. The microcavity type digital PCR system has an open characteristic and is suitable for various PCR reaction requirements.
In 2013, Life Technologies introduced a micro-cavity based QuantstudioTMA digital PCR chip of the 3D digital PCR system adopts silicon-based materials, and 20000 micropore arrays are processed on the surface of a silicon chip and used for dividing samples. When the digital PCR chip is used for detection, redundant samples exist on the digital PCR chip after the micropore array is filled with the samples, and the redundant samples influence the PCR reaction when being more, so that the detection precision is influenced. The silicon-based digital PCR chip is produced in batches by adopting micro-nano processing technologies such as ultraviolet lithography and dry etching, the manufacturing process is complex, the yield is low, the cost is high, and the detection cost of a sample is greatly increased; and the sample flux is lower, and the space for improving the performance is limited, thereby greatly limiting the application range of the sample.
Disclosure of Invention
The invention mainly aims to provide a digital PCR chip, a preparation method, a preparation device and a using method thereof, which are used for solving the problems of high manufacturing cost, low yield, low sample flux, low PCR reaction heat conduction efficiency and the like of the digital PCR chip in the prior art and reducing the influence of redundant samples on the detection precision.
In order to achieve the above object, the present invention provides a digital PCR chip comprising: the device comprises a substrate and a structural layer arranged above the substrate, wherein the structural layer is provided with at least one sample channel, each sample channel comprises a microporous structure with a plurality of micropores and an overflow cavity for containing redundant samples, and the overflow cavity is positioned on at least one side of the microporous structure.
Further, the structural layer has a hydrophilic structure to enhance the hydrophilicity of the microporous structure.
Further, the substrate is a flexible polymer film.
Further, the substrate is a PET film.
Further, the structural layer is ultraviolet nano-imprint glue.
Further, the structural layer is PEGDA.
Further, the volume of the overflow cavity is Vc, the volume of the micropores is Vo, the number of the micropores is N, the volume of the sample is V, wherein Vc is more than or equal to V-VoxN, and/or the depth of the micropores is Hr, the depth of the overflow cavity is Hc, wherein Hc is less than or equal to Hr, and/or the thickness H of the substrate is 0.03-0.5 mm, and/or the micropores are round holes, elliptical holes, conical holes or polygonal holes, and/or a plurality of micropores form a rectangular array or a hexagonal array.
The invention also provides a preparation method for preparing the digital PCR chip, which comprises the following steps: step S10: coating an imprinting adhesive on a substrate; step S20: impressing the impressing glue coated on the substrate through the forming mold to form a microporous structure and an overflow cavity; step S30: curing and molding the imprinted imprinting adhesive: step S40: and demolding the solidified imprinting glue.
Further, in step S20, under the pressing action of the supporting structure and the forming mold, the imprinting glue is filled onto the template structure of the forming mold, and/or the surface of the forming mold has a hydrophilic structure, and/or the forming mold is a forming wheel, and the supporting structure is a supporting pressing wheel, and/or before step S10, the method further includes: step S01: the substrate is placed on the unwinding roll and the free end thereof is wound on the winding roll to transfer the substrate, and/or, in step S30, ultraviolet light is irradiated to the substrate from the side of the substrate having no micro-porous structure.
The invention also provides a preparation device for implementing the preparation method, which comprises the following steps: a transfer structure for transferring the substrate; the coating structure is used for coating the imprinting glue on the substrate; the molding structure comprises a molding die, wherein the surface of the molding die is provided with a plurality of first bulges arranged at intervals and second bulges surrounding at least one side of the first bulges, and the first bulges and the second bulges are respectively used for imprinting a microporous structure and an overflow cavity; and the ultraviolet curing structure is arranged on one side of the forming structure and is used for curing and forming the imprinted imprinting glue.
Further, the surface of the forming die is provided with a hydrophilic structure, and/or the transmission structure comprises an unwinding roller and a winding roller which are arranged at intervals, the substrate is placed on the unwinding roller, the free end of the substrate is wound on the winding roller, and/or the forming structure further comprises a supporting structure, the supporting structure and the forming die are arranged in a manner of being close to each other left and right, and/or the ultraviolet curing structure is arranged below the forming die.
The invention also provides a using method of the digital PCR chip, which comprises the following steps: step S100: dropwise adding the sample on a digital PCR chip; step S200: the sample is filled into the microporous structure by rolling the roller, the redundant sample overflows into the overflow cavity, and the sample is sealed; step S300: and heating the sealed digital PCR chip to enable the sample to have PCR reaction.
Further, in step S200, a sealing tape is wound around a roller to seal the sample while pressing the sample, and in step S300, the upper surface and the lower surface of the sealed digital PCR chip are heated, respectively, or in step S200, a disposable film is wound around the roller, and after the sample is pressed, mineral oil is dropped to seal the sample, and in step S300, the lower surface of the sealed digital PCR chip is heated.
Furthermore, when the roller is wound with the sealing tape, the thickness Ht of the sealing tape is 0.025-0.1 mm.
The technical scheme of the invention has the following advantages: the multiple micropores in the micropore structure are used for dividing samples, the sample flux is high, and the adjustable range is large; the overflow cavity is used for containing redundant samples, so that the redundant samples can be stored in the overflow cavity, the PCR reaction is not influenced, and the detection precision is improved. The chip structure is thin, and the efficiency of PCR reaction can be improved.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 shows a schematic perspective view of a digital PCR chip provided in a first embodiment of the present invention;
FIG. 2 shows an exploded schematic view of the digital PCR chip of FIG. 1;
FIG. 3 shows a schematic top view of the digital PCR chip of FIG. 1;
FIG. 4 shows a schematic cross-sectional view of the A-A direction of the digital PCR chip of FIG. 3;
FIG. 5 shows an enlarged schematic diagram at B of the digital PCR chip of FIG. 4;
FIG. 6 is a perspective view of a digital PCR chip provided in a second embodiment of the present invention;
FIG. 7 shows a schematic top view of the digital PCR chip of FIG. 6;
FIG. 8 is a simplified schematic diagram of an apparatus for preparing a digital PCR chip according to the present invention;
FIG. 9 is a schematic diagram showing a structure of a dropping sample in a method of using the digital PCR chip provided in the first embodiment of the present invention;
FIG. 10 shows a schematic diagram of a structure of a squeeze sample in a method of using the digital PCR chip of FIG. 9;
FIG. 11 is a schematic diagram showing a structure of a sealed sample in a method of using the digital PCR chip of FIG. 9;
FIG. 12 is a schematic view showing a structure of an extrusion sample in a method for using a digital PCR chip provided in a second embodiment of the present invention.
Description of reference numerals:
11. a substrate; 12. a structural layer; 121. micropores; 122. an overflow chamber; 13. a sealing layer; 21. unwinding rollers; 22. coating the structure; 23. supporting the pinch roller; 24. a forming wheel; 25. an ultraviolet curing structure; 26. a wind-up roll; 30. a roller; 40. sealing the adhesive tape; 50. and (3) sampling.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the accompanying drawings, and it should be understood that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In the description of the present invention, it should be noted that the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc., indicate orientations or positional relationships based on the orientations or positional relationships shown in the drawings, and are only for convenience of description and simplicity of description, but do not indicate or imply that the device or element being referred to must have a particular orientation, be constructed and operated in a particular orientation, and thus, should not be construed as limiting the present invention. Furthermore, the terms "first," "second," and "third" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance.
In the description of the present invention, it should be noted that, unless otherwise explicitly specified or limited, the terms "mounted," "connected," and "connected" are to be construed broadly, e.g., as meaning either a fixed connection, a removable connection, or an integral connection; can be mechanically or electrically connected; they may be connected directly or indirectly through intervening media, or they may be interconnected between two elements. The specific meanings of the above terms in the present invention can be understood in specific cases to those skilled in the art.
In addition, the technical features involved in the different embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
Example one
As shown in fig. 1 and 2, the digital PCR chip of the present embodiment includes: a substrate 11 and a structural layer 12 arranged above the substrate 11, the structural layer 12 having a sample channel, each sample channel comprising a microporous structure having a plurality of micropores 121 and an overflow chamber 122 for holding excess sample, the overflow chamber 122 being located on at least one side of the microporous structure.
By applying the digital PCR chip of the embodiment, the plurality of micropores 121 in the micropore structure are used for dividing samples, the sample flux is high, and the adjustable range is large; the overflow cavity 122 is used for containing redundant samples, so that the redundant samples can be stored in the overflow cavity, the PCR reaction cannot be influenced, and the detection precision is improved. The chip structure is thin, and the efficiency of PCR reaction can be improved.
In this embodiment, the structural layer 12 has a hydrophilic structure to enhance the hydrophilicity of the microporous structure, so that the sample can be spread out on the structural layer to fill the microporous structure.
In this embodiment, the substrate 11 is a flexible polymer film, which has the advantages of low cost, high performance, high quality, high precision, and the like, and can realize rapid and batch production of chips. Specifically, the substrate 11 is a PET (polyethylene terephthalate) film. The PET film has the following advantages: 1. the PET film has excellent physical and mechanical properties in a large temperature range, the long-term working temperature is more than 100 ℃, the mechanical properties are slightly influenced by temperature change, and the requirement of PCR reaction on temperature is met. 2. The PET film is colorless and transparent, has high transmittance, and can be used for ultraviolet imprinting. 3. The PET film is nontoxic and tasteless and has good biocompatibility. Of course, the substrate may be a PC film, a PMMA film, or the like, but is not limited thereto.
According to the curing mode division, nano-imprinting can be divided into hot imprinting and ultraviolet imprinting, compared with the hot imprinting, the ultraviolet imprinting has lower requirements on pressure and temperature, the template structure and the substrate thermal expansion caused by heating can be avoided, and more accurate pattern transfer can be obtained. The invention adopts ultraviolet imprinting to realize pattern transfer, and the imprinting adhesive is directly contacted with a sample, so the imprinting adhesive is required to have good biocompatibility, specifically, the imprinting adhesive is an ultraviolet light cured high molecular polymer and is used as a structural layer, namely, the structural layer 12 is ultraviolet nano imprinting adhesive which has good biocompatibility and does not react with the sample, and the ultraviolet nano imprinting adhesive is used for transferring a template structure of a forming mold to a PET film to form a microcavity array so as to realize sample division. Preferably, the structural layer 12 is PEGDA (polyethylene glycol diacrylate) and the photosensitizer is 2-Hydroxy-2-methylprophenone (2-Hydroxy-2-methyl-1-phenyl-1-propanone).
In this embodiment, the surface modification treatment of the PET film can enhance the adhesion between the film and the imprint glue, and prevent the imprint glue from separating from the PET film substrate during the mold release process.
In this embodiment, the overflow cavity 122 is a U-shaped groove, i.e., the overflow cavity 122 surrounds a portion of the outside of the microporous structure. Of course, the shape of the overflow chamber is not limited thereto, and needs to be adjusted according to the shape of the microporous structure.
In the embodiment, the volume of the overflow cavity 122 is Vc, the volume of the micropores 121 is Vo, the number of the micropores 121 is N, and the volume of the sample 50 is V, wherein Vc is not less than V-Vo × N, so as to further prevent the overflow of the sample.
In this embodiment, the depth of the micro-hole 121 is Hr, and the depth of the overflow cavity 122 is Hc, if Hc > Hr, the thickness of the lower portion of the structure layer located under the micro-hole structure is increased, and the thickness of the whole chip is increased, which is not good for heat conduction. In order to improve the heat conduction effect, Hc is required to be less than or equal to Hr, so that the thickness of the part, positioned below the microporous structure, of the structural layer is thinner, the heat conduction is fast when the digital PCR chip is heated, the amplification time of a sample is shortened, and the depth of the microporous structure can be accurately controlled in the forming process. Specifically, Hr is 0.001 to 0.25 mm. Preferably, Hr is 0.03-0.1 mm.
In this embodiment, the substrate 11 is used as a carrier of the structural layer 12 for carrying the imprint resist, and the thickness H of the substrate 11 is 0.03-0.5 mm, so as to improve the mechanical strength of the chip. Preferably, the thickness H of the substrate 11 is 0.05 to 0.1 mm.
In the present embodiment, as shown in fig. 3 to 5, the micro-holes 121 are tapered holes. The taper angle of the micro-holes 121 is 0 to 60 degrees. Preferably, the taper angle of the micro-holes 121 is 0 ° to 30 °. Wherein, the taper hole can be a conical hole or a pyramid hole, etc. Of course, the micro-holes may be circular holes, elliptical holes, polygonal holes (such as triangular holes, rectangular holes, etc.), and the like.
In the present embodiment, the plurality of microwells 121 form a hexagonal array, increasing the number of microwells per unit area, and improving sample throughput. Of course, the plurality of microwells 121 may also form a rectangular array.
Example two
Fig. 6 and 7 show the structure of the second embodiment of the digital PCR chip of the present invention, which is different from the first embodiment in the number of sample channels, and in the second embodiment, the structural layer 12 has eight sample channels. Of course, the number of sample channels is not limited thereto.
The invention also provides a preparation method for preparing the digital PCR chip, which comprises the following steps:
step S10: coating an imprinting adhesive on a substrate;
step S20: imprinting the imprinting glue coated on the substrate through a molding die to form a micro-pore structure and an overflow cavity 122;
step S30: curing and molding the imprinted imprinting adhesive:
step S40: and demolding the solidified imprinting glue.
The preparation method has the following advantages:
1. because the consistency of the volume of the reaction unit influences the detection precision of the digital PCR, particularly for high-concentration samples, the nano-imprinting process has high precision and good consistency of the size of the microporous structure, the volume of the reaction unit can be accurately controlled, and the consistency of the volume of the reaction unit can be further improved, so that the detection precision of the digital PCR is improved.
2. The size of the sample flux is related to the dynamic range of the digital PCR detection, so that the sample flux is increased, the dynamic range of the digital PCR detection can be improved, the resolution of the nanoimprint process is high, more microporous structures can be imprinted on the surface of the film in unit area, the sample flux is greatly improved, and the dynamic range of the digital PCR detection is further improved.
3. The nano-imprinting process can imprint nano-to micron-sized structures, has a large structural size range, can greatly improve the application range of the digital PCR chip, and has a large performance improvement space.
4. The digital PCR chip is manufactured by adopting the nanoimprint technology, complex photoetching and etching technologies are not needed, the technology is simple, the cost is low, and efficient and batch production can be realized.
In this embodiment, the substrate is a flexible polymer film, and the flexible polymer film has the advantages of low cost, high efficiency, high quality, high precision, and the like, and can realize rapid and batch production of chips. Specifically, the substrate is a PET (polyethylene terephthalate) film. The PET film has the following advantages: 1. the PET film has excellent physical and mechanical properties in a large temperature range, the long-term working temperature is more than 100 ℃, the mechanical properties are slightly influenced by temperature change, and the requirement of PCR reaction on temperature is met. 2. The PET film is colorless and transparent, has high transmittance, and can be used for ultraviolet imprinting. 3. The PET film is nontoxic and tasteless and has good biocompatibility. Of course, the substrate may be a PC film, a PMMA film, or the like, but is not limited thereto.
In the embodiment, the imprinting adhesive is ultraviolet nano-imprinting adhesive which has good biocompatibility and does not react with the sample, and the ultraviolet nano-imprinting adhesive is used for transferring the template structure of the forming mold to the PET film to form a microcavity array, so that sample division is realized. Specifically, the imprinting adhesive is PEGDA (polyethylene glycol diacrylate), and the photosensitizer is 2-Hydroxy-2-methylprophenone (2-Hydroxy-2-methyl-1-phenyl-1-propanone).
In this embodiment, in step S20, the imprint paste is filled onto the template structure of the molding die under the pressing action of the support structure and the molding die. The template structure of the forming mold is complementary to the pore structure and the overflow cavity 122 of the structural layer, so that the pore structure and the overflow cavity 122 are formed on the imprinting glue. Of course, the above preparation method can prepare a digital PCR chip without an overflow chamber, and is not limited thereto.
In this embodiment, the surface of the forming mold has a hydrophilic structure, so that the template structure of the forming mold can be imprinted on the film, and the hydrophilic structure can be imprinted on the film, and then an inherent hydrophilic layer is formed on the surface of the microporous structure, so that the surface hydrophilic treatment of the digital PCR chip is not required, and the sample can be directly added on the digital PCR chip for use.
In this embodiment, as shown in fig. 8, the forming mold is a forming wheel 24, and the supporting structure is a supporting pressing wheel 23, that is, a roll-to-roll nano-imprinting process is adopted, so that the large-area micro-porous structure can be rapidly produced in batch, and the method has the advantages of low cost, high efficiency, high quality, high precision and the like, and is very suitable for imprinting the flexible thin film. Through the distance between adjustment support pinch roller 23 and the shaping wheel 24, can improve the homogeneity of glue film, the thickness that the accurate control impression was glued ensures that the impression is glued fully to fill the template structure on, reduces the thickness that the impression was glued simultaneously. Specifically, the supporting pinch roller 23 is a rubber pinch roller, which does not damage the film and protects the film.
In the present embodiment, the molding wheel 24 includes a wheel body and a metal mold plate structure provided on an outer circumferential surface of the wheel body. Specifically, the surface of the metal mold plate structure is provided with a plurality of first bulges arranged at intervals and second bulges surrounding the outer sides of parts of the plurality of first bulges, the first bulges are used for stamping the micropores, and the second bulges are used for stamping the overflow cavities. Of course, a roll-to-plate nanoimprint process may also be employed, where the molding mold is a molding plate that includes a fixing plate and a metal mold plate structure disposed on a lower surface of the fixing plate.
Preferably, the metal mold plate structure is bonded on the wheel body, so that the metal mold plate structure is convenient to replace. Of course, the metal mold plate structure may be fixed on the wheel body by other fixing methods, or the wheel body and the metal mold plate structure may be integrally formed.
In this embodiment, before step S10, the method further includes: step S01: the substrate is placed on an unwinding roller 21 and the free end of the substrate is wound on a winding roller 26, the unwinding roller 21 and the winding roller 26 are used for conveying the substrate, and the winding roller 26 winds the embossed chips. And cutting the rolled digital PCR chips into digital PCR chips with different lengths according to requirements, and further obtaining the digital PCR chips with different sample channel numbers, namely obtaining the digital PCR chips with different specifications.
In step S30, the ultraviolet light irradiates the substrate from the side of the substrate without the micro-porous structure, i.e. the substrate is cured and formed by means of ultraviolet curing, the ultraviolet curing needs to meet a certain energy requirement, and the energy depends on the power of the ultraviolet light and the exposure time. The exposure time can be controlled by adjusting the transmission speed of the PET film, the power of ultraviolet light, the thickness of the imprinting glue and the transmission speed of the film are optimized, and the production efficiency is improved while the imprinting glue is solidified. And the power of ultraviolet light, the transmission rate of the PET film and the thickness of the imprinting glue are adjusted, so that the rapid curing of the imprinting glue is realized.
The following describes the process flow of the preparation method of the digital PCR chip:
unreeling: the PET film roll is placed on the unwinding roller, is drawn out for conveying, the position and the angle of the PET film roll are adjusted, the film is guaranteed not to bend or fold in the moving process, and then static on the surface of the PET film is removed.
Coating: and after static electricity is removed, the PET film is conveyed to a coating structure according to a specified route, and nano-imprint glue is dripped to coat the nano-imprint glue on the surface of the film.
Molding: and conveying the PET film coated with the stamping glue to a forming structure, and fully filling the stamping glue into the metal mold structure of the forming wheel under the extrusion of the supporting pressing wheel and the forming wheel.
And (3) curing: and (3) conveying the PET film to an ultraviolet curing structure, irradiating the imprinting glue by ultraviolet light, and rapidly curing the imprinting glue so as to transfer the metal template structure of the forming wheel to the PET film and form a microporous structure and an overflow cavity.
Winding: and demolding and separating the solidified imprinting glue from the molding wheel to finish demolding, and then conveying the demolded PET film to a winding roller to realize winding and finish the preparation of the digital PCR chip.
The present invention also provides a manufacturing apparatus for implementing the above manufacturing method, as shown in fig. 8, the manufacturing apparatus including: a transfer structure for transferring the substrate, a coating structure 22, a forming structure and an ultraviolet curing structure 25; the coating structure 22 is used for coating the imprinting glue on the substrate; the molding structure comprises a molding die, the surface of the molding die is provided with a plurality of first bulges arranged at intervals and second bulges surrounding at least one side of the plurality of first bulges, and the plurality of first bulges and the second bulges are respectively used for imprinting the microporous structure and the overflow cavity 122; the ultraviolet curing structure 25 is arranged on one side of the molding structure and is used for curing and molding the imprinted imprinting glue.
In this embodiment, the surface of the forming mold has a hydrophilic structure, so that the template structure of the forming mold can be imprinted on the film, and the hydrophilic structure can be imprinted on the film, and then an inherent hydrophilic layer is formed on the surface of the microporous structure, so that the surface hydrophilic treatment of the digital PCR chip is not required, and the sample can be directly added on the digital PCR chip for use.
In this embodiment, the transmission structure includes the unwinding roller 21 and the winding roller 26 that the interval set up, and the base is placed on the unwinding roller 21 and its free end twines on the winding roller 26, simplifies the transmission structure, and then makes the structure of preparation facilities simpler.
In this embodiment, the forming structure further comprises a supporting structure, the supporting structure and the forming mold are arranged in a manner of being close to each other left and right, and the template structure of the forming mold is transferred to the imprinting glue under the action of the supporting structure and the forming mold. Specifically, the forming mold is a forming wheel 24, the supporting structure is a supporting pressing wheel 23, namely, a roll-to-roll nano-imprinting process is adopted, so that the large-area micro-pore structure can be rapidly produced in batches, and the method has the advantages of low cost, high efficiency, high quality, high precision and the like, and is very suitable for imprinting of the flexible thin film. Through the distance between adjustment support pinch roller 23 and the shaping wheel 24, can improve the homogeneity of glue film, the thickness that the accurate control impression was glued ensures that the impression is glued fully to fill the template structure on, reduces the thickness that the impression was glued simultaneously. Specifically, the supporting pinch roller 23 is a rubber pinch roller, which does not damage the film and protects the film.
In the present embodiment, the forming wheel 24 includes a wheel body and a metal mold plate structure disposed on the outer circumferential surface of the wheel body, and the surface of the metal mold plate structure has a plurality of first protrusions and second protrusions. Of course, a roll-to-plate nanoimprint process may also be employed, where the molding mold is a molding plate that includes a fixing plate and a metal mold plate structure disposed on a lower surface of the fixing plate.
Preferably, the metal mold plate structure is bonded on the wheel body, so that the metal mold plate structure is convenient to replace. Of course, the metal mold plate structure may be fixed on the wheel body by other fixing methods, or the wheel body and the metal mold plate structure may be integrally formed.
In this embodiment, the unwinding roller 21 is disposed on one side of the supporting pinch roller 23 far away from the forming wheel 24, the winding roller 26 is disposed above the forming wheel 24, and the free end of the substrate passes through the space between the supporting pinch roller 23 and the forming wheel 24 and then is wound on the winding roller 26, so as to simplify the structure of the manufacturing device.
In the present embodiment, the ultraviolet curing structure 25 is disposed below the forming mold, so as to cure the imprinted imprinting glue. The ultraviolet curing structure is used for realizing curing molding of the nano imprinting glue, and finally the microporous structure is formed.
In this embodiment, the coating structure 22 is used for coating the nanoimprint lithography glue on the surface of the PET film, and the coating structure adopts a glue dripping mode to coat the nanoimprint lithography glue, so that the glue dripping amount is controlled, and overflow glue is prevented from occurring and the supporting pressure roller is prevented from being polluted. In particular, the glue drip of the coating structure 22 is arranged above the portion of the substrate tangent to both the supporting pinch wheel 23 and the forming wheel 24. Of course, the coating structure may be in other locations.
Fig. 9 to 11 show a method of using the digital PCR chip of the present invention, the method comprising the steps of:
step S100: dropping the sample 50 onto the digital PCR chip;
step S200: the sample 50 is filled into the micro-porous structure by pressing the sample 50 by the rolling of the roller 30 and the excessive sample 50 overflows into the overflow chamber 122, and the sample 50 is sealed;
step S300: the sealed digital PCR chip is heated to allow PCR reaction of the sample 50.
The sample is extruded through the rolling of the rolling shaft, the sample can be fully filled into the microporous structure, and meanwhile, redundant samples are extruded to the overflow cavity, so that the operation is simple and convenient, and the detection efficiency is improved. Sealing the sample 50 prevents evaporation of the sample during amplification while avoiding sample cross-contamination.
In this embodiment, in step S200, the sealing tape 40 is wound around the roller 30, and the sample 50 is sealed while the sample 50 is pressed, so that the operation is simple. After the roller 30 is rolled from one side of the chip to the other side, the sealing tape 40 is cut off to facilitate sealing of the following chip, improving efficiency. The sealing tape 40 serves as a sealing layer for sealing the micro well array, wherein the sealing tape 40 has biocompatibility and does not react with the sample. Specifically, the sealing tape 40 is a single-sided tape, the working temperature of the single-sided tape is greater than 100 ℃, and the single-sided tape is colorless and transparent, so that the reaction unit can be conveniently observed in real time in the PCR reaction process. The single-sided tape serves as the sealing layer 13, and the overall thickness of the chip is greatly reduced. In step S300, the upper surface and the lower surface of the sealed digital PCR chip are heated respectively to form a sandwich structure, thereby improving the heat conduction effect, shortening the amplification time of the sample, and greatly improving the efficiency of the PCR reaction.
In the present embodiment, the thickness Ht of the sealing tape 40 is 0.025-0.1 mm. Preferably, the thickness Ht of the sealing tape 40 is 0.025-0.05 mm.
Before step S100, the method further includes: the digital PCR chip is fixed on a metal heating flat plate, wherein the structural layer faces upwards. And a metal heating flat plate is also placed above the sealed digital PCR chip, and then the upper surface and the lower surface of the digital PCR chip are respectively heated. If the template structure of the forming mould has no hydrophilic structure, the surface of the digital PCR chip is subjected to hydrophilic treatment to enhance the hydrophilicity of the microporous structure, so that a sample can be conveniently spread on the structural layer and enter the microporous structure; then the digital PCR chip is fixed on a metal heating plate. Wherein the hydrophilic treatment is: and (3) placing the digital PCR chip into oxygen plasma treatment equipment, and treating the surface of the structure by using oxygen plasma to form silicon hydroxyl on the surface of the microporous structure so as to enhance the hydrophilicity.
FIG. 12 shows another method of using the digital PCR chip of the present invention, which includes the steps of:
fixing a digital PCR chip on a metal heating plate, wherein a structural layer faces upwards;
dropping the sample 50 onto the digital PCR chip;
the sample 50 is extruded by the rolling of the roller 30, so that the sample 50 is filled into the microporous structure, the excessive sample 50 overflows to the overflow cavity 122, and the mineral oil is dripped to seal the sample 50;
the lower surface of the sealed digital PCR chip is heated to allow the PCR reaction of the sample 50 to occur.
In which a disposable film is wound around the roller 30, and after the roller 30 is rolled from one side of the chip to the other side, the film is cut and the cut film is discarded.
The mineral oil has low viscosity and density and good fluidity, is convenient for sealing the microporous structure, and does not react with the sample and the impression gum. Wherein the working temperature of the mineral oil is more than 100 ℃. The mineral oil is colorless and transparent, so that the reaction unit can be conveniently observed in real time in the PCR reaction process.
From the above description, it can be seen that the above-described embodiments of the present invention achieve the following technical effects:
1. the digital PCR chip takes the PET film and the stamping glue as chip base materials, has good physical and mechanical properties, temperature characteristics and biocompatibility, has low material cost and is convenient for batch production. And use PET film and impression to glue as the chip substrate, the whole thickness of chip can reduce by a wide margin, and the heat conduction effect obtains promoting, can show like this and shorten the required time of sample PCR reaction, improves detection efficiency.
2. The digital PCR chip uses a PET film as a substrate, biocompatible imprinting glue is coated on the surface of the PET film, a microporous structure and an overflow cavity are imprinted on the imprinting glue, a microcavity structure is used for dividing samples, the overflow cavity is used for containing redundant samples, and the digital PCR chip is prepared by adopting a nano-imprinting process and has the following advantages: (1) the processing precision of the nano-imprinting process is high, and the consistency of the micropore structure can be ensured; (2) the nano-imprinting process has high resolution, more micro-cavity array structures can be imprinted in unit area, and the sample flux is greatly improved; (3) the nano-scale to micron-scale array structure can be imprinted by adopting a nano-imprinting process, the adjustable range of the structure size is large, and the application range of the digital PCR chip can be greatly improved; (4) more digital PCR chips can be manufactured in unit time, the production efficiency is high, and the cost is greatly reduced.
3. The digital PCR chip is flexible and convenient in use, and the digital PCR chips with different specifications can be cut according to actual requirements.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.
Claims (14)
1. A digital PCR chip, comprising: a substrate (11) and a structural layer (12) arranged above said substrate (11), said structural layer (12) having at least one sample channel, each of said sample channels comprising a microporous structure having a plurality of micropores (121) and an overflow chamber (122) for containing excess sample, said overflow chamber (122) being located on at least one side of said microporous structure.
2. The digital PCR chip according to claim 1, wherein the structural layer (12) has a hydrophilic structure to enhance the hydrophilicity of the microporous structure.
3. The digital PCR chip according to claim 1, wherein the substrate (11) is a flexible polymer film.
4. The digital PCR chip according to claim 3, wherein the substrate (11) is a PET film.
5. The digital PCR chip according to claim 1, wherein the structural layer (12) is an ultraviolet nanoimprint paste.
6. The digital PCR chip of claim 5, wherein the structural layer (12) is PEGDA.
7. The digital PCR chip according to claim 1, wherein the volume of the overflow cavity (122) is Vc, the volume of the microwell (121) is Vo, the number of the microwells (121) is N, and the volume of the sample (50) is V, wherein Vc is more than or equal to V-VoxN, and/or the depth of the microwell (121) is Hr, the depth of the overflow cavity (122) is Hc, wherein Hc is less than or equal to Hr, and/or the thickness H of the substrate (11) is 0.03-0.5 mm, and/or the microwell (121) is a circular hole, an elliptical hole, a conical hole or a polygonal hole, and/or a plurality of the microwells (121) form a rectangular array or a hexagonal array.
8. A method for preparing the digital PCR chip of any one of claims 1 to 7, comprising:
step S10: coating an imprinting adhesive on a substrate;
step S20: imprinting the imprinting glue coated on the substrate through a forming mold to form a micropore structure and an overflow cavity (122);
step S30: curing and molding the imprinted imprinting adhesive:
step S40: and demolding the solidified imprinting glue.
9. The method for preparing according to claim 8, wherein in step S20, the impression compound is filled onto the template structure of the forming mold under the pressing action of the supporting structure and the forming mold, and/or the surface of the forming mold has hydrophilic structure, and/or the forming mold is a forming wheel (24), the supporting structure is a supporting pressing wheel (23), and/or before step S10, the method further comprises: step S01: placing a substrate on an unwinding roll (21) and winding a free end thereof on a winding roll (26) to transfer the substrate, and/or, in the step S30, irradiating the substrate with ultraviolet light from a side of the substrate not having the microporous structure.
10. A production apparatus for carrying out the production method according to any one of claims 8 to 9, comprising:
a transfer structure for transferring the substrate;
a coating structure (22) for coating an imprint resist on the substrate;
the molding structure comprises a molding die, wherein the surface of the molding die is provided with a plurality of first bulges arranged at intervals and second bulges surrounding at least one side of the first bulges, and the first bulges and the second bulges are respectively used for imprinting a microporous structure and an overflow cavity (122);
and the ultraviolet curing structure (25) is arranged on one side of the forming structure and is used for curing and forming the imprinted imprinting glue.
11. The production apparatus according to claim 10, wherein the surface of the forming die has a hydrophilic structure, and/or wherein the transport structure comprises an unwinding roller (21) and a winding roller (26) arranged at intervals, the substrate being placed on the unwinding roller (21) and the free end thereof being wound on the winding roller (26), and/or wherein the forming structure further comprises a support structure arranged right and left next to the forming die, and/or wherein the uv-curing structure (25) is arranged below the forming die.
12. A method of using the digital PCR chip of any one of claims 1 to 7, comprising the steps of:
step S100: dropping a sample (50) onto the digital PCR chip;
step S200: pressing the sample (50) by the rolling of the roller (30) to fill the sample (50) into the microporous structure and overflow the sample (50) in excess into an overflow chamber (122), and sealing the sample (50);
step S300: and heating the sealed digital PCR chip to enable the sample (50) to generate PCR reaction.
13. Use according to claim 12,
in the step S200, a sealing tape 40 is wound on the roller 30, the sample 50 is sealed while the sample 50 is pressed,
in the step S300, the upper surface and the lower surface of the sealed digital PCR chip are respectively heated,
or,
in the step S200, a disposable film is wound on the roller (30), mineral oil is dropped to seal the sample (50) after the sample (50) is extruded,
in step S300, the lower surface of the sealed digital PCR chip is heated.
14. The use method according to claim 13, wherein the sealing tape (40) has a thickness Ht of 0.025 to 0.1mm when the sealing tape (40) is wound around the roller (30).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910070932.6A CN109797096B (en) | 2019-01-25 | 2019-01-25 | Digital PCR chip and preparation method, preparation device and use method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910070932.6A CN109797096B (en) | 2019-01-25 | 2019-01-25 | Digital PCR chip and preparation method, preparation device and use method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109797096A true CN109797096A (en) | 2019-05-24 |
| CN109797096B CN109797096B (en) | 2024-03-12 |
Family
ID=66558813
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910070932.6A Active CN109797096B (en) | 2019-01-25 | 2019-01-25 | Digital PCR chip and preparation method, preparation device and use method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109797096B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112415366A (en) * | 2020-11-18 | 2021-02-26 | 浙江清华柔性电子技术研究院 | Structure for constructing test microenvironment |
| CN112547147A (en) * | 2020-11-23 | 2021-03-26 | 武汉世纪康敏生物科技有限公司 | Immunodetection chip and preparation method thereof |
| CN112892627A (en) * | 2021-02-05 | 2021-06-04 | 浙江大学 | Photocuring micro-fluidic chip based on elastic support body and preparation method and application thereof |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105543064A (en) * | 2015-12-29 | 2016-05-04 | 西安交通大学 | Digital PCR chip and using method thereof |
| CN106755420A (en) * | 2015-12-31 | 2017-05-31 | 中国科学院上海微系统与信息技术研究所 | Digital pcr chip and method based on surfactant-modified PDMS |
| CN107262170A (en) * | 2017-07-03 | 2017-10-20 | 重庆大学 | A kind of multiple digital pcr chip and its application method |
| CN108373969A (en) * | 2018-01-11 | 2018-08-07 | 中国科学院上海微系统与信息技术研究所 | A kind of digital pcr chip and preparation method thereof and application method |
| CN108624478A (en) * | 2018-07-12 | 2018-10-09 | 西安交通大学 | A kind of digital pcr chip and its application method based on hydrogel |
| CN109182092A (en) * | 2018-10-19 | 2019-01-11 | 苏州德思普生物科技有限公司 | A kind of micro-fluidic chip and its application for detection of nucleic acids |
-
2019
- 2019-01-25 CN CN201910070932.6A patent/CN109797096B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105543064A (en) * | 2015-12-29 | 2016-05-04 | 西安交通大学 | Digital PCR chip and using method thereof |
| CN106755420A (en) * | 2015-12-31 | 2017-05-31 | 中国科学院上海微系统与信息技术研究所 | Digital pcr chip and method based on surfactant-modified PDMS |
| CN107262170A (en) * | 2017-07-03 | 2017-10-20 | 重庆大学 | A kind of multiple digital pcr chip and its application method |
| CN108373969A (en) * | 2018-01-11 | 2018-08-07 | 中国科学院上海微系统与信息技术研究所 | A kind of digital pcr chip and preparation method thereof and application method |
| CN108624478A (en) * | 2018-07-12 | 2018-10-09 | 西安交通大学 | A kind of digital pcr chip and its application method based on hydrogel |
| CN109182092A (en) * | 2018-10-19 | 2019-01-11 | 苏州德思普生物科技有限公司 | A kind of micro-fluidic chip and its application for detection of nucleic acids |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112415366A (en) * | 2020-11-18 | 2021-02-26 | 浙江清华柔性电子技术研究院 | Structure for constructing test microenvironment |
| CN112415366B (en) * | 2020-11-18 | 2023-03-10 | 浙江清华柔性电子技术研究院 | Structure for constructing test microenvironment |
| CN112547147A (en) * | 2020-11-23 | 2021-03-26 | 武汉世纪康敏生物科技有限公司 | Immunodetection chip and preparation method thereof |
| CN112892627A (en) * | 2021-02-05 | 2021-06-04 | 浙江大学 | Photocuring micro-fluidic chip based on elastic support body and preparation method and application thereof |
| CN112892627B (en) * | 2021-02-05 | 2022-04-05 | 浙江大学 | Photocuring micro-fluidic chip based on elastic support body and preparation method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109797096B (en) | 2024-03-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109797096B (en) | Digital PCR chip and preparation method, preparation device and use method thereof | |
| Anderson et al. | Fabrication of topologically complex three-dimensional microfluidic systems in PDMS by rapid prototyping | |
| Tanaka et al. | Microcasting with agarose gel via degassed polydimethylsiloxane molds for repellency-guided cell patterning | |
| Weibel et al. | Combining microscience and neurobiology | |
| CN112543678B (en) | Patterned microfluidic devices and methods of making the same | |
| US20210101316A1 (en) | Nanoimprinting by using soft mold and resist spreading | |
| JP2016511931A (en) | How to make micro contact imprint | |
| KR101565835B1 (en) | Fabrication method of replication mold, fine structures using the same and its applications thereof. | |
| CN113426500A (en) | Preparation method of nanofluidic chip based on nano-corrugated structure | |
| CN209778857U (en) | Digital PCR chip and preparation device thereof | |
| US10124332B2 (en) | Reversible bonding of microfluidic channels using dry adhesives | |
| CN108545692B (en) | Method for manufacturing microfluidic chip with inner wall of channel coated with parylene | |
| Biswas et al. | Soft lithography using nectar droplets | |
| CN104708800A (en) | Soft imprinting method for manufacturing micro-nano structure in cycloalkene polymer micro-fluidic chip | |
| Zhong et al. | Hot roller embossing of multi-dimensional microstructures using elastomeric molds | |
| CN110262186B (en) | Nano-imprinting mold, nano-imprinting device and nano-imprinting method | |
| CN108008599B (en) | Method and device for three-dimensional curved surface nanoscale imprinting and mold preparation method | |
| CN110862926A (en) | Multilayer paper chip based on microfluidic technology and construction method thereof | |
| WO2023109661A1 (en) | Manufacturing method for micro-nano composite structure | |
| WO2023142479A1 (en) | Inverted gas expansion forming method for inclined micropore array | |
| TW201932279A (en) | Method for embossing at least one microstructure or nanostructure with an embossing die | |
| KR102218428B1 (en) | Micropatterning method via microcontact printing and degas-driven flow guided patterning, and self-assembled monolayer prepared thereby | |
| KR20230145460A (en) | Microfluidic system consisting of folded foil and manufacturing method | |
| US20220195129A1 (en) | Method for producing microparticles | |
| CN113337451A (en) | Accurate patterning method for single cells in multi-shear force microfluidic chip |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |