CN110227563B - Evaporation-proof sealing method for PDMS (polydimethylsiloxane) micro-fluidic chip and PDMS micro-fluidic chip - Google Patents
Evaporation-proof sealing method for PDMS (polydimethylsiloxane) micro-fluidic chip and PDMS micro-fluidic chip Download PDFInfo
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- CN110227563B CN110227563B CN201910393044.8A CN201910393044A CN110227563B CN 110227563 B CN110227563 B CN 110227563B CN 201910393044 A CN201910393044 A CN 201910393044A CN 110227563 B CN110227563 B CN 110227563B
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/502707—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the manufacture of the container or its components
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L7/00—Heating or cooling apparatus; Heat insulating devices
- B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0887—Laminated structure
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/12—Specific details about materials
Abstract
The invention provides an anti-evaporation sealing method for a PDMS micro-fluidic chip and the PDMS micro-fluidic chip. The sealing method comprises forming a photoresist layer on a clean glass sheet; attaching a mask with a microfluidic chip pattern structure to the surface of the photoresist layer, and exposing and developing to obtain a template; flatly paving a template on the surface of a glass substrate, pasting an adhesive tape along the edge of the template, wherein the thickness of the adhesive tape is larger than that of the template, fumigating the glass substrate, pouring a paraffin oil dispersion liquid of polydimethylsiloxane, vacuumizing, pasting a first transparent glass slide, and solidifying and separating the first transparent glass slide to obtain a chip; aligning and attaching the vacuum plasma processing chip and the second transparent glass slide with the hole; luer connector with sealing cover. The micro-fluidic chip obtained by the invention can effectively prevent evaporation, thereby avoiding the problems of shrinkage, volatilization, fusion, deformation and the like generated in the temperature change process of the micro-reaction unit.
Description
Technical Field
The invention belongs to the technical field of microfluidic chips, and particularly relates to an anti-evaporation sealing method for a PDMS microfluidic chip and the PDMS microfluidic chip.
Background
Polymerase Chain Reaction (PCR) has a very important position in the field of molecular biology, and has been developed into a conventional experimental technique. With continuous improvement from the original general PCR, reverse transcription PCR, multiplex PCR, nested PCR, immuno-PCR, etc. are gradually derived. And also gradually evolve from initial qualitative analysis to quantitative analysis. With the continuous development of over ten years, real time PCR (qPCR) is becoming the most common technical means for gene analysis and widely used in disease diagnosis due to its advantages of high sensitivity, strong specificity and accurate quantification. However, qPCR can only be "relatively quantitative" due to a variety of factors. Therefore, there is a need for a new technique that can "absolutely quantify" to solve the problem of limited sensitivity and accuracy in nucleic acid quantification. Based on such demands, digital PCR (digital PCR, abbreviated as dPCR) technology has come to work. The technology does not need to rely on any standard or external standard, and can achieve the purpose of absolute quantification only by directly counting the positive signals of the single lattice nucleic acid molecules, so that the sensitivity and the precision are extremely superior, the technology can be used for quantitative analysis only by a very small sample amount, and the characteristics enable the digital PCR technology to be rapidly and widely applied in various fields, and a Micro-reaction chamber/orifice plate (Micro-chamber), a Micro-fluidic chip (Microfluidic chip) and a Micro-droplet PCR system (ddPCR) are developed.
Microfluidic chips are generally composed of two layers of Polydimethylsiloxane (PDMS) film and a bottom layer of cover glass, so as to form a corresponding flow layer and a control layer. The PDMS membrane is processed by a soft lithography technology, so that a crisscross gas channel and a liquid channel are formed, and fluid can be quickly divided into parallel reaction units by corresponding valve control. PDMS material is a high molecular organic silicon compound, and has the characteristics of optical transparency, hydrophobicity, waterproofness and the like. The polymer material is widely applied to the fields of micro-fluidic control and the like due to low cost, simple use, strong biocompatibility, transparency and air permeability. However, due to the air permeability of the PDMS material, the sealing performance of the digital PCR chip made of the PDMS material is often difficult, and the micro-reaction units in the digital PCR chip are shrunk, volatilized, fused, deformed, and the like during the temperature variation process, which causes the non-uniformity of the micro-reaction units and even the reaction failure, and thus the application of the PDMS material in the digital PCR microfluidic chip is also greatly limited.
Disclosure of Invention
Aiming at the problems of nonuniform reaction and even failure caused by phenomena of shrinkage, volatilization, deformation and the like of a micro-reaction unit of the conventional digital PCR micro-fluidic chip during temperature change, the invention provides an anti-evaporation sealing method for a PDMS micro-fluidic chip.
Further, the invention also provides the PDMS microfluidic chip obtained by the sealing method.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a PDMS micro-fluidic chip evaporation prevention sealing method comprises the following steps:
step S01, coating photoresist on the surface of a clean glass sheet, and then carrying out heating curing treatment to form a photoresist layer laminated on the surface of the glass sheet;
s02, attaching a mask with a microfluidic chip pattern structure to the surface of the photoresist layer, and carrying out exposure and development treatment to obtain a template with the microfluidic chip pattern structure;
s03, flatly paving the template on the surface of a glass substrate, pasting a layer of adhesive tape on the surface of the glass substrate along the periphery of the edge of the template, enabling the thickness of the adhesive tape to be larger than that of the template, fumigating the glass substrate attached with the adhesive tape by adopting an isolation liquid, forming an isolation layer on the surface of the template, pouring a paraffin oil dispersion liquid of polydimethylsiloxane onto the surface of the template, vacuumizing to enable the paraffin oil solution of the polydimethylsiloxane to be uniformly distributed on the surface of the template, pasting a first transparent glass slide, and curing the first transparent glass slide to obtain a chip with a belt structure;
s04, separating the chip with the structure from the template;
s05, placing the chip with the belt structure and the second transparent glass slide with the holes under a vacuum condition for plasma treatment, and then aligning, attaching and heating the chip with the belt structure and the second transparent glass slide with the holes;
and S06, coating a layer of glue on the surface of the second transparent glass slide with the hole, adhering the luer connector with the sealing cover to the surface of the second transparent glass slide, aligning the luer connector with the hole of the second transparent glass slide to form an inlet and an outlet of the microfluidic chip, and heating to obtain the PDMS microfluidic chip.
Correspondingly, the PDMS microfluidic chip comprises a first transparent glass slide, a PDMS film layer laminated on the surface of the first transparent glass slide, and a second transparent glass slide laminated on the surface of the PDMS film layer;
the surface of the second transparent glass slide is provided with a plurality of through holes, and the through holes are connected with Ruhr joints with sealing covers.
The invention has the technical effects that:
compared with the prior art, the anti-evaporation sealing method of the PDMS microfluidic chip provided by the invention has the advantages that the PDMS film layer is attached to the surface of the transparent glass slide, then plasma treatment is carried out, and then the PDMS film layer is attached to the second transparent glass slide, so that an airtight transparent structure layer is formed on the surface of the PDMS film layer, the contact area of the PDMS film layer and air is reduced, and luer connectors with sealing covers are connected at an inlet and an outlet, so that the anti-evaporation effect is achieved, and the problems of shrinkage, volatilization, fusion, deformation and the like of a micro-reaction unit in a digital PCR chip in the temperature change process are solved.
According to the PDMS microfluidic chip provided by the invention, the airtight transparent structure layers are formed on the two opposite surfaces of the PDMS film layer, and the Ruhr joints with the sealing covers are connected at the inlet and the outlet, so that the evaporation prevention effect is effectively achieved, and the problems of shrinkage, volatilization, fusion, deformation and the like of a micro-reaction unit in the temperature change process are avoided.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to these drawings without creative efforts.
FIG. 1 is a schematic view of a clean glass plate provided by the anti-evaporation sealing method for PDMS microfluidic chips according to the present invention;
FIG. 2 is a schematic view of a photoresist layer formed on the surface of a clean glass plate by the anti-evaporation sealing method for the PDMS microfluidic chip according to the present invention;
FIG. 3 is a schematic diagram of a process of attaching a mask having a microfluidic chip pattern structure to the surface of a photoresist layer by using the anti-evaporation sealing method for the PDMS microfluidic chip provided by the invention;
fig. 4 is a schematic structural diagram formed by flatly paving the obtained template on the surface of the glass substrate and pasting a layer of adhesive tape on the surface of the glass substrate along the periphery of the edge of the template by the anti-evaporation sealing method for the PDMS microfluidic chip provided by the invention;
fig. 5 is a schematic structural diagram of a PDMS film layer formed by pouring a PDMS paraffin oil solution on the template and curing the PDMS paraffin oil solution according to the anti-evaporation sealing method for the PDMS microfluidic chip provided by the present invention;
FIG. 6 is a schematic structural diagram of a PDMS film layer surface coated with a first transparent glass slide according to the sealing method for preventing evaporation of the PDMS microfluidic chip provided by the present invention;
fig. 7 is a schematic structural diagram formed by performing plasma treatment on a first transparent glass slide and a second transparent glass slide and bonding the first transparent glass slide and the second transparent glass slide according to the anti-evaporation sealing method for the PDMS microfluidic chip provided by the present invention;
fig. 8 is a schematic structural diagram of a PDMS microfluidic chip obtained by connecting a luer connector of a sealing cover to the surface of a second transparent glass slide according to the sealing method for preventing evaporation of the PDMS microfluidic chip provided by the present invention;
wherein, 1-glass sheet; 2-photoresist (template); 3-masking; 4-a glass substrate; 5-adhesive tape; 6-PDMS film layer; 7-a first transparent slide; 8-second transparent slide, 81-through hole; 9-luer fitting with sealed cap.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Referring to fig. 1 to 7, the present invention provides an anti-evaporation sealing method for a PDMS microfluidic chip, including the following steps:
a clean and dry glass sheet 1 is provided, as shown in detail in fig. 1. The glass sheet 1 should be clean and dry to avoid the influence of residual oil stains and stains on the surface on the processing effect.
A photoresist layer 2 is coated on the surface of the glass sheet 1 by spin coating, as shown in detail in fig. 2. The photoresist forming the photoresist layer 2 may be a positive photoresist or a negative photoresist. Specifically, the positive photoresist is selected from any one of polymethyl methacrylate (PMMA) and o-diazonaphthoquinone-phenolic resin (DQN); the negative photoresist SU8 photoresist.
Specifically, the photoresist is dropped on the surface of the glass sheet 1 to perform spin coating treatment, the obtained solution is left to stand for about 5 minutes to obtain a photoresist layer 2, and then the photoresist layer 2 is subjected to heating curing treatment at 50-70 ℃, for example, curing can be performed at 55 ℃, 60 ℃ and 65 ℃ to obtain the cured photoresist layer 2. Naturally cooling to room temperature, attaching the mask 3 with the microfluidic chip pattern structure to the surface of the photoresist layer 2, and performing exposure and development treatment to obtain the template 2 (the number is the same as that of the photoresist layer) formed by the photoresist layer 2, as shown in fig. 3 in detail.
During the exposure process, ultraviolet rays are used for exposure treatment, ethyl lactate or tetramethylammonium hydroxide (TMAH) is used for development, and after the development is finished, isopropanol is used for judging whether the development is completely finished. The template 2 formed by the photoresist layer 2 has a microfluidic chip pattern structure thereon.
Taking a clean and dry glass substrate 4, ensuring that the size of the glass substrate 4 is larger than that of the template 2, then flatly paving the template 2 on the surface of the glass substrate 4, attaching a layer of adhesive tape 5 on the surface of the glass substrate 4 along the peripheral edge of the template 2, and ensuring that the thickness of the adhesive tape 5 is larger than that of the template 2, so as to be beneficial to forming the PDMS film layer 6. Fumigating the glass substrate 4 attached with the adhesive tape 5 by using an isolation liquid for 5-10 min, and forming an isolation layer on the surface of the template 2 by fumigating to prevent the subsequent PDMS film 6 from being adhered to the template 2, as shown in detail in FIG. 4.
The isolation liquid is dichlorodimethylsilane, and an isolation layer formed by the isolation liquid can separate the PDMS film layer 6 from the template 2 in the subsequent process. The formed isolation layer is a dichlorodimethylsilane film layer.
The paraffin oil dispersion liquid of Polydimethylsiloxane (PDMS) is poured on the surface of the template 2, and then vacuum treatment is carried out, so that the PDMS is completely filled in the pattern structure of the microfluidic chip of the template 2. The vacuum-pumping time is generally 20-40 min, for example, vacuum-pumping treatment can be performed for 25min, 30min, 35min to form the PDMS film layer 6. And then, adhering the first transparent glass slide 7 to the surface of the PDMS film layer 6, and heating to bond the first transparent glass slide 7 and the PDMS film layer 6 into a whole, wherein the heating temperature is 50-70 ℃, the heating time is 1-2 h, and the details are shown in FIG. 6.
Preferably, the PDMS film layer 6 is formed to have a microfluidic chip pattern, and the thickness of the PDMS film layer 6 is not greater than 0.25 mm. If the thickness of the PDMS film layer 6 is too thick, the contact area with air is increased, and the finished product is easy to evaporate in the reaction process, thereby causing the problems of deformation and the like.
And separating the PDMS film layer 6 attached with the first transparent glass slide 7 from the template 2 to obtain a chip with a structure, wherein the chip with the structure comprises the first transparent glass slide 7 and the PDMS film layer 6.
And respectively placing the chip with the structure and the second transparent glass slide 8 with the hole under a vacuum condition for plasma treatment so as to facilitate the two to be attached, wherein the second transparent glass slide 8 is provided with a plurality of through holes 81, and the through holes 81 are mainly used for subsequently forming an inlet and an outlet of the microfluidic chip, such as forming two inlets and one outlet. After plasma treatment, the second transparent glass slide 8 and the chip with the structure are aligned and attached, and are heated to be tightly bonded. During the attaching process, the second transparent glass slide 8 is bonded to the PDMS film layer 6, so as to form a "sandwich" structure of the first transparent glass slide 7-PDMS film layer 6-second transparent glass slide 8, as shown in fig. 7 in detail.
After a layer of glue is coated on the surface of the second transparent glass slide 8, the Ruhr joint 9 with the sealing cover is bonded with the through hole 81, and heating treatment is carried out at the heating temperature of 50-70 ℃, so that the Ruhr joint 9 with the sealing cover and the second transparent glass slide 8 form a good bonding effect.
According to the sealing method provided by the invention, the PDMS film layer is attached to the surface of the transparent glass slide, then plasma treatment is carried out, and then the PDMS film layer is attached to the second transparent glass slide, so that an airtight transparent structure layer is formed on the surface of the PDMS film layer, the contact area of the PDMS film layer and air is reduced as much as possible, and luer connectors with sealing covers are connected at an inlet and an outlet, so that the effect of evaporation resistance is achieved, and the problems of shrinkage, volatilization, fusion, deformation and the like of a micro-reaction unit in a digital PCR chip in the temperature change process are solved.
Therefore, the invention provides the PDMS microfluidic chip obtained by the sealing method. Specifically, as shown in fig. 8, the PDMS microfluidic chip includes a first transparent glass slide 7, a PDMS film layer 6 laminated on a surface of the first transparent glass slide 7, and a second transparent glass slide 8 laminated on a surface of the PDMS film layer 6;
the surface of the second transparent glass slide 8 is provided with a plurality of through holes 81, and the through holes 81 are connected with a luer connector 9 with a sealing cover. And a luer joint 9 with a sealing cover is communicated with the PDMS film layer 6 to form an inlet and an outlet of the PDMS microfluidic chip.
According to the PDMS microfluidic chip, the airtight transparent structure layers are formed on the two opposite surfaces of the PDMS film layer, and the Ruhr joints with the sealing covers are bonded at the inlet and the outlet, so that the evaporation prevention effect is effectively achieved, and the problems of shrinkage, volatilization, fusion, deformation and the like in the temperature change process of the micro-reaction unit are avoided.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (8)
1. A PDMS micro-fluidic chip evaporation prevention sealing method is characterized by comprising the following steps:
step S01, coating photoresist on the surface of a clean glass sheet, and then carrying out heating curing treatment to form a photoresist layer laminated on the surface of the glass sheet;
s02, attaching a mask with a microfluidic chip pattern structure to the surface of the photoresist layer, and carrying out exposure and development treatment to obtain a template with the microfluidic chip pattern structure;
s03, flatly paving the template on the surface of a glass substrate, pasting a layer of adhesive tape on the surface of the glass substrate along the periphery of the edge of the template, enabling the thickness of the adhesive tape to be larger than that of the template, fumigating the glass substrate attached with the adhesive tape by adopting an isolation liquid, forming an isolation layer on the surface of the template, pouring a paraffin oil dispersion liquid of polydimethylsiloxane onto the surface of the template, vacuumizing to enable the paraffin oil dispersion liquid of the polydimethylsiloxane to be uniformly distributed on the surface of the template, pasting a first transparent glass slide, and curing the first transparent glass slide to obtain a chip with a belt structure;
s04, separating the chip with the structure from the template;
s05, placing the chip with the belt structure and the second transparent glass slide with the holes under a vacuum condition for plasma treatment, and then aligning, attaching and heating the chip with the belt structure and the second transparent glass slide with the holes;
s06, coating a layer of glue on the surface of the second transparent glass slide with the hole, adhering a luer connector with a sealing cover on the surface of the second transparent glass slide, aligning the luer connector with the hole of the second transparent glass slide to form an inlet and an outlet of the microfluidic chip, and heating to obtain the PDMS microfluidic chip;
wherein the thickness of the PDMS layer formed by solidifying the paraffin oil dispersion liquid of the polydimethylsiloxane is not more than 0.25 mm.
2. The method for sealing a PDMS microfluidic chip against evaporation of claim 1, wherein the photoresist is a positive photoresist or a negative photoresist.
3. A PDMS microfluidic chip evaporation-resistant sealing method according to claim 2, wherein said positive photoresist is selected from any one of poly methyl methacrylate, o-diazonaphthoquinone-phenol resin; the negative photoresist is selected from SU8 photoresist.
4. The method for sealing a PDMS microfluidic chip of claim 1 against evaporation, wherein the plasma treatment time is 2-5 min.
5. The method for sealing a PDMS microfluidic chip against evaporation of claim 1, wherein the temperature of the heating treatment in step S05 is 50-70 ℃.
6. The method for sealing a PDMS microfluidic chip against evaporation according to claim 1, wherein the curing process of step S03 is performed by heat preservation at 50-70 ℃ for 1-3 h.
7. The method for sealing a PDMS microfluidic chip against evaporation according to claim 1, wherein the developing solution is any one of ethyl lactate and tetramethylammonium hydroxide.
8. A PDMS microfluidic chip manufactured by the sealing method according to any one of claims 1 to 7, comprising a first transparent glass slide, a PDMS film layer laminated on a surface of the first transparent glass slide, and a second transparent glass slide laminated on a surface of the PDMS film layer;
the surface of the second transparent glass slide is provided with a plurality of holes, and the holes are connected with Ruhr joints with sealing covers.
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| CN107328744B (en) * | 2017-05-17 | 2021-01-01 | 中国科学院上海微系统与信息技术研究所 | Micro-fluidic chip for detecting tumor marker based on uniformly distributed microspheres and application method thereof |
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| CN101813695A (en) * | 2009-12-30 | 2010-08-25 | 复旦大学 | Microfludic chip for rapid detection of microcystins and preparation method thereof |
| CN102240534A (en) * | 2011-05-20 | 2011-11-16 | 中国科学院化学研究所 | Method for manufacturing three-dimensional micromixer microfluidic chip |
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Inventor after: Xu Tao Inventor after: Chen Songfeng Inventor before: Chen Songfeng |
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