CN111269832A - Preparation method of circular truncated cone-shaped micro-pit array chip based on one-time exposure - Google Patents
Preparation method of circular truncated cone-shaped micro-pit array chip based on one-time exposure Download PDFInfo
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- 238000002360 preparation method Methods 0.000 title claims abstract description 11
- 238000000034 method Methods 0.000 claims abstract description 30
- 229920002120 photoresistant polymer Polymers 0.000 claims abstract description 23
- 239000000758 substrate Substances 0.000 claims abstract description 17
- 239000004205 dimethyl polysiloxane Substances 0.000 claims abstract description 14
- 229920000435 poly(dimethylsiloxane) Polymers 0.000 claims abstract description 14
- 239000011521 glass Substances 0.000 claims abstract description 7
- 229910052710 silicon Inorganic materials 0.000 claims abstract description 7
- 239000010703 silicon Substances 0.000 claims abstract description 7
- 235000013870 dimethyl polysiloxane Nutrition 0.000 claims description 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 description 12
- 238000004987 plasma desorption mass spectroscopy Methods 0.000 claims description 12
- 238000010438 heat treatment Methods 0.000 claims description 11
- LZCLXQDLBQLTDK-UHFFFAOYSA-N ethyl 2-hydroxypropanoate Chemical compound CCOC(=O)C(C)O LZCLXQDLBQLTDK-UHFFFAOYSA-N 0.000 claims description 8
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 claims description 6
- 229940116333 ethyl lactate Drugs 0.000 claims description 4
- 239000003999 initiator Substances 0.000 claims description 4
- 239000000178 monomer Substances 0.000 claims description 4
- 238000001816 cooling Methods 0.000 claims description 3
- 238000005520 cutting process Methods 0.000 claims description 2
- 230000015572 biosynthetic process Effects 0.000 abstract description 2
- -1 polydimethylsiloxane Polymers 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 16
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 3
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 3
- 210000002744 extracellular matrix Anatomy 0.000 description 3
- 239000004005 microsphere Substances 0.000 description 3
- 238000004528 spin coating Methods 0.000 description 3
- 238000004220 aggregation Methods 0.000 description 2
- 230000002776 aggregation Effects 0.000 description 2
- 239000008346 aqueous phase Substances 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- 210000002242 embryoid body Anatomy 0.000 description 2
- 210000004263 induced pluripotent stem cell Anatomy 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000000465 moulding Methods 0.000 description 2
- 239000012071 phase Substances 0.000 description 2
- 230000006978 adaptation Effects 0.000 description 1
- 230000019552 anatomical structure morphogenesis Effects 0.000 description 1
- 238000003491 array Methods 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 230000008614 cellular interaction Effects 0.000 description 1
- 230000005754 cellular signaling Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
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- 238000001727 in vivo Methods 0.000 description 1
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- 239000007791 liquid phase Substances 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 238000002715 modification method Methods 0.000 description 1
- 238000004264 monolayer culture Methods 0.000 description 1
- 239000003921 oil Substances 0.000 description 1
- 238000001259 photo etching Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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- 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
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Abstract
The invention relates to a preparation method of a circular truncated cone-shaped micro-pit array chip based on one-time exposure. The preparation method mainly comprises the steps of rotating a rotatable platform at a constant speed and inclining at a certain angle, exposing photoresist coated on a substrate (glass/silicon wafer) in a rotating mode on the rotatable platform, developing, hardening, and finally performing back mold by PDMS (polydimethylsiloxane), so that the uniform round table-shaped micro-pit array chip is prepared. The chip can be used for cell fixation, cell balling culture, pseudoembryo formation and the like. Compared with the existing micro-pit preparation method, the method is simple to operate, only needs one-time exposure, does not need complex operation, has high success rate and small batch difference, and can realize larger depth-to-width ratio.
Description
Technical Field
The invention relates to the field of fine processing and manufacturing of a chip micron structure and the like, and relates to a preparation method of a circular truncated cone-shaped micro-pit array chip based on one-time exposure, which provides a new means for pseudoembryo formation and cell 3D culture.
Background
Most cells in an organism exhibit a three-dimensional structure, with each cell interacting with neighboring cells and extracellular matrix (ECM) components. Cell/cell and cell/ECM interactions are key factors for morphogenesis, cell signaling, cell viability and functional maintenance. However, most conventional cell culture methods are based on 2D culture systems. Although they are well established, these 2D monolayer cultures lack tight 3D cell/cell interactions and therefore do not reflect the true in vivo environment, leading to a loss of tissue specificity and increasing uncertainty in their effectiveness. The methods for forming 3D cell aggregates commonly used by scholars at present mainly include a pendant drop method, a substrate group modification method, a low adhesion micro-pit method, a material wrapping method, and the like. Among them, the micropit method has the characteristics of easy operation and generalization, is distinct from many methods, and is widely used.
The micro-pit chip can be prepared by various methods, such as using frozen beads as a template and then performing reverse molding, deforming an elastic membrane by using vacuum and then performing reverse molding, and a pneumatic method. These methods are complicated to operate, and the stability of methods such as an ice-bead template method and a pneumatic method is not high enough, so that the formed micro-pit arrays are easy to have different sizes and large batch differences, and the bottom surfaces are all arc-shaped (the cell convergence capacity is weak), so that the high aspect ratio (the low aspect ratio is easy to cause the cells to come out of the pits under the fluid) cannot be realized.
In conclusion, a simple method for preparing a micro-pit array with high consistency, strong cell aggregation capability and large aspect ratio is very significant.
Disclosure of Invention
The invention aims to develop a simple method for preparing a micro-pit array with high consistency, strong cell aggregation capability and large depth-to-width ratio.
The invention provides a method for preparing a circular truncated cone-shaped micro-pit array chip based on one-time exposure. The method comprises the following steps:
spin-coating photoresist with a certain thickness on a glass or silicon wafer substrate by using a photoresist homogenizer, and placing a heated photoresist substrate 3 on a rotatable platform 4;
and secondly, placing the glass or silicon chip substrate on a rotatable customized platform with a certain inclination angle, and adjusting the inclination of the platform support 5.
Selecting an exposure template;
step four, the rotatable platform 4 rotates at a constant speed, and the vertical parallel light source exposes;
step five, developing to obtain a circular truncated cone-shaped micro-pit array chip template;
pouring uncured PDMS on the truncated cone-shaped micro-pit array chip template obtained in the fifth step, removing bubbles and curing at high temperature;
step seven: and 6, cutting off the PDMS in the sixth step to obtain the truncated cone-shaped micro-pit array chip.
Preferably, the photoresist substrate 3 is made of glass or silicon wafer.
In the first step, the thickness of the photoresist on the surface of the photoresist substrate 3 is as follows: 100 to 800 μm.
In the first step, the heating temperature of the photoresist substrate 3 is 95 ℃, and the heating time is 0.5-5 h.
In the second step, the inclination of the platform support 5 is adjusted within the following range: 20 to 60 degrees.
And a film plate with a circular hole array is printed on the exposure template in the third step.
The rotating speed of the rotating platform 4 in the fourth step is 2-20 degrees/s, the number of rotating turns is 1-3 turns, and the exposure time is 20s-2 min.
And further, developing by using ethyl lactate in the fifth step, heating in an oven at 180 ℃ for 2h, and naturally cooling.
In the step five, the ratio of the initiator to the monomers in the uncured PDMS is 1: 8-10 ℃, the curing temperature is 80-120 ℃, and the curing time is 0.5-1 h.
The final pits of the circular truncated cone-shaped micro-pit array chip are circular truncated cone-shaped, and the area of the bottom is smaller than that of the top.
A film plate printed with a circular array (the size of a circle is the size of the bottom of the last small pit) is used as an exposure template, vertical parallel light is used for exposure, and a rotary platform rotates at a constant speed during exposure. The exposure time is determined by the photoresist thickness and is 2 to 3 times longer than the vertical exposure time for the same thickness photoresist to ensure that all exposed areas are cured. The larger the inclination angle of the supporting platform is, the smaller the angle between the side edge of the circular truncated cone and the bottom surface is, and the larger the opening at the upper end of the pit of the finally obtained PDMS pit array chip is. The speed of the rotating platform should not be too fast. The exposure effect is better when the exposure device rotates for 1-3 circles completely within the exposure time.
The invention provides a preparation method of a circular truncated cone-shaped micro-pit array based on one-time exposure, which is photoetching.
The key point of the method for preparing the circular truncated cone-shaped micro-pit array based on one-time exposure is that the inclined rotatable platform 4 is adopted to carry out vertical parallel light exposure on the photoresist.
The preparation method of the circular truncated cone-shaped micro-pit array based on one-time exposure, provided by the invention, has the advantages that the exposure time is determined by the thickness of the photoresist, and the exposure is generally carried out for 20s-2min to ensure that the exposed part is solidified. The larger the inclination angle of the inclined platform is, the smaller the angle between the side edge of the circular truncated cone and the ground is, and the larger the opening at the upper end of the pit of the PDMS pit array chip is finally obtained. The speed of the rotary platform is not too fast, and the number of rotation turns is 1-3.
The invention has the following advantages:
(1) the method is simple and easy to operate.
(2) The method is stable, the batch difference is small, and the size of the small pit array is uniform.
(3) A higher aspect ratio can be achieved.
(4) The small pits are in a round table shape, and the cell gathering capacity is strong. Under the condition of proper light intensity, the small pits close to the cone can be realized.
The small pit size is controllable.
Drawings
In order to more clearly illustrate the technical scheme of the invention, the drawings used in the technical scheme description are simply introduced.
FIG. 1 is a schematic view of a production apparatus of the present invention;
FIG. 2 is a schematic diagram of a preparation method of the method;
the mask plate comprises a mask plate 1 with a circular hole array pattern, photoresist 2, a photoresist substrate 3, a rotatable platform 4 and a platform support 5; 2a is the exposure rotated by 0 degree, and 2b is the exposure rotated by 180 degree.
Detailed Description
The present invention is further described with reference to the following specific examples, but the scope of the present invention is not limited by the examples, and if one skilled in the art makes some insubstantial modifications and adaptations to the present invention based on the above disclosure, the present invention still falls within the scope of the present invention.
Example 1
And spin-coating 800-micron photoresist on a glass or silicon wafer substrate by using a photoresist homogenizer. After heating at 95 ℃ for a period of 5h, the substrate was placed on a custom made rotatable platform (4) with an inclination of 20 °. A film plate printed with a circular array with the radius of 50 microns is used as an exposure template, vertical parallel light is used for exposure, a rotary platform rotates at a constant speed during exposure, and the rotating speed is 6 degrees/s. Exposing for 2min, developing with ethyl lactate, heating in an oven at 180 deg.C for 2h, and naturally cooling. The ratio of initiator to monomer was 8: pouring the uncured PDMS of 1 on a template, removing bubbles, and heating in an oven at 80 ℃ for 30min for curing. And finally, stripping PDMS to obtain the uniform round table shaped micro-pit array chip (the diameter of the bottom of the small pit is one hundred micrometers, the diameter of the top of the small pit is about 550 micrometers, and the depth of the small pit is 800 micrometers).
The prepared micro-pit array is autoclaved, dried, washed for three times by PBS and rinsed once by the human induced pluripotent stem cell culture medium. The human induced pluripotent stem cells digested into single cells are added on the micro-pit array after being resuspended by using a culture medium, and the cells can be self-assembled into embryoid bodies after 4 hours due to the convergence of gravity and the micro-pits, so that 3D culture can be carried out, and each embryoid body can be conveniently operated and observed.
Example 2
And spin-coating photoresist with the thickness of 100 microns on a glass or silicon wafer substrate by using a photoresist homogenizer. After heating at 95 ℃ for 30min, the substrate was placed on a rotatable custom platform with an inclination of 60 °. A film plate printed with a fifty-micron radius circular array is used as an exposure template, vertical parallel light is used for exposure, a rotary platform rotates at a constant speed during exposure, the rotating speed is 18 degrees/s, the exposure time is 20s, ethyl lactate is used for development, the film plate is heated in an oven at 180 ℃ for 2h, and finally the film plate is cooled naturally. The ratio of initiator to monomer was 10: pouring the uncured PDMS of 1 on a template, removing bubbles, and heating in an oven at 120 ℃ for 1h for curing. And finally, stripping PDMS to obtain the uniform round table shaped micro-pit array chip (the diameter of the bottom of the small pit is one hundred micrometers, the diameter of the top of the small pit is about 350 micrometers, and the depth of the small pit is 100 micrometers).
After the crater array with 350 micron diameter at the top is prepared, a certain amount of water phase microspheres (300 micron diameter) dispersed in the oil phase are dripped on the crater array. There is only one aqueous phase microsphere at most in one pit. Different liquid phase reactions can be carried out by utilizing each aqueous phase microsphere and continuous tracking observation is carried out.
Claims (10)
1. A preparation method based on a one-time exposure round table-shaped micro-pit array chip is characterized by comprising the following steps: the preparation method comprises the following specific steps:
step one, placing a heated photoresist substrate (3) on a rotatable platform (4);
secondly, adjusting the inclination of the platform support (5);
selecting an exposure template;
step four, the rotatable platform (4) rotates at a constant speed, and the vertical parallel light source exposes;
step five, developing to obtain a circular truncated cone-shaped micro-pit array chip template;
pouring uncured PDMS on the truncated cone-shaped micro-pit array chip template obtained in the fifth step, removing bubbles and curing at high temperature;
step seven: and 6, cutting off the PDMS in the sixth step to obtain the truncated cone-shaped micro-pit array chip.
2. The method for preparing a truncated cone-shaped micro-pit array chip according to claim 1, wherein: the photoresist substrate (3) is made of glass or silicon chip.
3. The method for preparing a truncated cone-shaped micro-pit array chip according to claim 1, wherein: in the first step, the thickness of the photoresist on the surface of the photoresist substrate (3) is as follows: 100 to 800 μm.
4. The method for preparing a truncated cone-shaped micro-pit array chip according to claim 1, wherein: in the first step, the heating temperature of the photoresist substrate (3) is 95 ℃, and the heating time is 0.5-5 h.
5. The method for preparing a truncated cone-shaped micro-pit array chip according to claim 1, wherein: in the second step, the inclination of the platform support (5) is adjusted within the range that: 20 to 60 degrees.
6. The method for preparing a truncated cone-shaped micro-pit array chip according to claim 1, wherein: and a film plate with a circular hole array is printed on the exposure template in the third step.
7. The method for preparing a truncated cone-shaped micro-pit array chip according to claim 1, wherein: the rotating speed of the rotating platform (4) in the fourth step is 2-20 degrees/s, the number of rotating turns is 1-3 turns, and the exposure time is 20s-2 min.
8. The method for preparing a truncated cone-shaped micro-pit array chip according to claim 1, wherein: and fifthly, developing by using ethyl lactate, heating in an oven at 180 ℃ for 2h, and naturally cooling.
9. The method for preparing a truncated cone-shaped micro-pit array chip according to claim 1, wherein: in the step five, the ratio of the initiator to the monomers in the uncured PDMS is 1: 8-10 ℃, the curing temperature is 80-120 ℃, and the curing time is 0.5-1 h.
10. The method for preparing a truncated cone-shaped micro-pit array chip according to claim 1, wherein: the final pits of the circular truncated cone-shaped micro-pit array chip are circular truncated cone-shaped, and the area of the bottom is smaller than that of the top.
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| CN201811477477.3A CN111269832A (en) | 2018-12-05 | 2018-12-05 | Preparation method of circular truncated cone-shaped micro-pit array chip based on one-time exposure |
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050206866A1 (en) * | 2003-12-26 | 2005-09-22 | Fuji Photo Film Co., Ltd. | Exposure method and exposure system |
| CN101281365A (en) * | 2007-04-04 | 2008-10-08 | 韩国科学技术院 | Material pattern, and mold, metal thin-film pattern, metal pattern using thereof, and methods of forming the same |
| CN104599864A (en) * | 2015-01-22 | 2015-05-06 | 太原理工大学 | Oblique photoetching method capable of increasing specific surface area of MEMS super-capacitor electrode |
| CN104701020A (en) * | 2015-03-20 | 2015-06-10 | 太原理工大学 | Preparation method of SU-8 photoresist-based three-dimensional microelectrode |
| CN108795751A (en) * | 2017-04-28 | 2018-11-13 | 中国科学院大连化学物理研究所 | One kind " funnel " sample three-dimensional cell aggregation culture chip and preparation method thereof |
| CN108795752A (en) * | 2017-04-28 | 2018-11-13 | 中国科学院大连化学物理研究所 | One kind " pyramid " sample three-dimensional cell aggregation culture chip and preparation method thereof |
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- 2018-12-05 CN CN201811477477.3A patent/CN111269832A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050206866A1 (en) * | 2003-12-26 | 2005-09-22 | Fuji Photo Film Co., Ltd. | Exposure method and exposure system |
| CN101281365A (en) * | 2007-04-04 | 2008-10-08 | 韩国科学技术院 | Material pattern, and mold, metal thin-film pattern, metal pattern using thereof, and methods of forming the same |
| CN104599864A (en) * | 2015-01-22 | 2015-05-06 | 太原理工大学 | Oblique photoetching method capable of increasing specific surface area of MEMS super-capacitor electrode |
| CN104701020A (en) * | 2015-03-20 | 2015-06-10 | 太原理工大学 | Preparation method of SU-8 photoresist-based three-dimensional microelectrode |
| CN108795751A (en) * | 2017-04-28 | 2018-11-13 | 中国科学院大连化学物理研究所 | One kind " funnel " sample three-dimensional cell aggregation culture chip and preparation method thereof |
| CN108795752A (en) * | 2017-04-28 | 2018-11-13 | 中国科学院大连化学物理研究所 | One kind " pyramid " sample three-dimensional cell aggregation culture chip and preparation method thereof |
Non-Patent Citations (1)
| Title |
|---|
| MANHEE HAN ET AL.: ""3D microfabrication with inclined/rotated UV lithography"", 《SENSORS AND ACTUATORS A》, vol. 111, no. 1, 1 March 2004 (2004-03-01), pages 8 - 9, XP004489316, DOI: 10.1016/j.sna.2003.10.006 * |
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Application publication date: 20200612 |