CN110862926A - A multi-layer paper chip based on microfluidic technology and its construction method - Google Patents

Abstract

The invention discloses a multi-layer paper chip based on a microfluidic technology and a construction method thereof, wherein the paper chip is mainly formed by sealing three layers of structures and comprises a lower layer structure, a middle layer structure and an upper layer structure; the lower layer structure is composed of a lower layer liquid inlet (1), a lower layer liquid pool (3) and a lower layer liquid outlet (2), the middle layer structure is composed of two middle layer holes and a middle layer hydrophilic and hydrophobic area (6), the upper layer structure is composed of an upper layer liquid inlet (7), an upper layer liquid pool (9) and an upper layer liquid outlet (8), and the construction method comprises the steps of patterning qualitative filter paper and integrating a microfluidic channel. The invention has simple manufacture, low cost, portability and good reproducibility, and is hopeful to become one of microenvironment equipment for single cell culture and co-culture of various cells.

Description

A multi-layer paper chip based on microfluidic technology and its construction method

technical field

The invention relates to a photolithography patterning technology and a microfluidic technology, in particular to a multi-layer paper chip based on the microfluidic technology and a construction method thereof.

Background technique

The paper chip uses paper as the base material to replace the traditional microfluidic chip with glass, silicon, polymer materials, etc. as the base. It is a device formed by patterning various microfluidic channels on paper. Its biggest feature is The liquid flow is driven by capillary action, and the introduction and reaction of the sample can be completed without external force driving. The material selection of the paper chip, so far, depends on whatman filter paper, nitrocellulose membrane, cellulose carbonate membrane and other materials with small pore size and uniform distribution, and the cost is relatively high. The present invention adopts ordinary qualitative filter paper, which is cheap and easy to obtain.

The two-dimensional paper chips produced by photolithography, paraffin printing, inkjet printing, drawing, laser processing, etc. have also become mature in the production of paper chips. The present invention adopts photolithography, and the patterned paper has higher hydrophilicity and hydrophobicity. Resolution, the requirements for equipment are relatively low, which avoids the low resolution of paraffin printing and the requirements for equipment such as laser, inkjet, and drawing. Moreover, two-dimensional paper chips cannot achieve high-throughput, multi-target detection. The three-dimensional paper chip prepared by the integrated method is a new type of paper chip developed in recent years. It breaks through this limitation and has the characteristics of miniaturization, integration, portability and multi-functionality. The network structure realizes the simultaneous analysis of multiple components and achieves the purpose of high-throughput and rapid detection.

At present, the methods for preparing three-dimensional paper chips include double-sided tape multi-layer stacking method and origami method. The double-sided tape multi-layer stacking method has a single form, and the adhesion between papers is through tape. Long-term storage will lead to the weakening of the adhesive force of the glue. Affecting the chip structure and subsequent applications, the origami method needs to consider the mirror image problem of the paper, the design is complicated, and the operation is inconvenient. The present invention adopts common qualitative filter paper, combined with photolithography patterning technology to prepare two-dimensional paper chips with uniformity, low cost and high resolution, and integrates light transmission, air permeability and good biocompatibility on the basis of two-dimensional paper chips. A simple, portable, low-sample, high-throughput, and reproducible three-dimensional paper chip was prepared by using oxygen and other irreversible sealing treatments to improve the capillary action of the hydrophilic region.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a multi-layer paper chip based on microfluidic technology and its construction method, which integrates PDMS microstructure, adopts photoresist infiltration technology, photolithography technology and microfluidic technology to integrate simple, portable, and sample size. A three-dimensional paper chip with less, high throughput and good reproducibility, the invention can be used for 3D culture microcarriers of cells, cell microenvironment simulation, drug sustained release, etc., and has good application value in the fields of medicine and biology.

A multi-layer paper chip based on microfluidic technology of the present invention is mainly formed by sealing a three-layer structure, and a multi-layer paper chip based on microfluidic technology is characterized in that: the paper chip is mainly sealed by a three-layer structure connected, including the lower layer structure, the middle layer structure and the upper layer structure;

The lower layer structure is composed of a lower layer liquid inlet, a lower layer liquid pool, and a lower layer liquid outlet, and the lower layer liquid pool is communicated with the lower layer liquid inlet and the lower layer liquid outlet;

The middle layer structure is composed of two middle layer holes and the middle layer hydrophilic and hydrophobic area, and the middle layer hydrophilic and hydrophobic area is not connected with the middle layer hole;

The upper layer structure is composed of the upper layer liquid inlet, the upper layer liquid pool, and the upper layer liquid outlet; the upper layer liquid pool is communicated with the upper layer liquid inlet and the upper layer liquid outlet;

The lower-layer liquid inlet communicates with the middle-layer hole 1, and the lower-layer liquid inlet communicates with the middle-layer hole 2;

The channel is a straight channel or a curved channel,

The channel is single channel or multi-channel,

The hydrophilic and hydrophobic regions of the intermediate layer are composed of a single circle or a plurality of patterns.

The paper core is formed by irreversible sealing of upper, middle and lower layers, the material of the upper layer and the lower layer are both light-transmitting and breathable PDMS polymers, and the material of the middle layer is a patterned qualitative filter paper.

The middle layer filter paper of the paper chip is patterned with a certain dilution ratio of SU8 glue (2025) to have obvious hydrophilic and hydrophobic regions, wherein the patterned SU8 glue (2025) is diluted with cyclopentanone, and the volume ratio of SU8 glue to cyclopentanone 1:1-1:5.

The invention provides a multi-layer paper chip based on microfluidic technology and a construction method thereof. The basic process is as follows:

(1) Patterned qualitative filter paper

After modifying the glass piece with trimethylchlorosilane vapor, bake it at 50℃-95℃ for a while, then throw a layer of PDMS, heat to solidify the PDMS, put a certain specification of qualitative filter paper on the PDMS, and use a syringe to drop a certain amount of dilution on the paper Proportion of SU8 glue (2025) to be flattened and the filter paper soaked with SU8 glue, defoamed in a vacuum drying oven, pre-baked at 60 ℃-95 ℃, squeezed with a glass sheet with PDMS layer, cooled naturally, exposed, Post-bake, develop after natural cooling, blow-dry, and dry at 50°C-95°C for 5-30min, to obtain a patterned paper in the middle layer, which is ready for use;

The trimethylchlorosilane steam modification time is 1-10min, the thickness of the PDMS on the glass sheet is 50-200 μm, the amount of SU8 glue dropped on the qualitative filter paper is 0.6-1.3mL, and the defoaming temperature range of the vacuum drying oven is 40 μm. ℃-70℃;

The patterns on the filter paper can be diversified (such as a structure formed by circular intervals, a straight channel structure, a serpentine structure, etc.), the development time is 20-50min, and the treatment time such as oxygen is 1-3min;

(2) Integrated microfluidic channel

Dewater the glass sheet for 0.5-2h, oxygen and other surface purification treatment for 1-3min, throw a certain thickness of SU8 glue, pre-bake, natural cooling, and then partially expose, post-bake, develop, harden the film at 90℃-180℃, and naturally cool the back side The template of SU8 was obtained. In order to make it hydrophobic, it was modified with trimethylchlorosilane, and then reversed with PDMS. After curing at 60°C-95°C, a PDMS chip containing a microfluidic channel structure can be obtained. Treated with oxygen, etc. The upper and lower layers of PDMS with the structure, the two sides of the middle layer patterned paper prepared in step (1) are also treated with oxygen and the like, and then the upper and lower layers of PDMS with channels and the middle layer patterned paper are irreversibly sealed, 60 The integrated paper chip is flat and well-sealed by extruding with copper plate at ℃-95℃, that is, a three-dimensional paper chip with integrated microfluidic channel is obtained;

The thickness of the SU8 glue is 50-500 μm, and the PDMS structure with microfluidic channels can be diversified, such as a single straight channel, a serpentine channel, a plurality of parallel channels, etc.;

The oxygen treatment time for the upper and lower PDMS layers with the structure is 0.5-3 min, and the double-sided oxygen treatment time for the paper patterned in the middle layer is 1-5 min.

The chip of the invention is simple to manufacture, cheap, portable, and has good reproducibility, making it promising to be one of the microenvironmental devices for single cell culture and co-culture of multiple cells.

Description of drawings

1 is a schematic diagram of the overall structure of a three-dimensional paper chip integrated with a microfluidic channel in Case 1 of the present invention;

2a, b, and c are respectively schematic diagrams of the lower layer, middle layer and upper layer of the three-dimensional paper chip with integrated microfluidic channel in Case 1 of the present invention;

Figure 3 is a scanning electron microscope characterization diagram of the intermediate layer of the integrated paper chip, which confirms that the area of SU8 and paper is clearly divided and that SU8 is completely saturated with filter paper under experimental conditions;

Figure 4 is a schematic diagram of the process of integrating a microfluidic channel;

Figure 5 is a physical diagram of a new multi-layer paper chip based on microfluidic technology;

FIG. 6 is a partial scanning electron microscope characterization image of the middle layer of the integrated paper chip in Example 2. FIG.

7 is a schematic diagram of the overall structure of a three-dimensional paper chip integrated with a microfluidic channel in Case 3 of the present invention;

8a, b, and c are respectively schematic diagrams of the lower layer, middle layer and upper layer of the three-dimensional paper chip with integrated microfluidic channel in Case 3 of the present invention;

Among them: 1 is the lower liquid inlet, 2 is the lower liquid outlet, 3 is the lower liquid pool, 4 is the middle layer hole 1, 5 is the middle layer hole 2, 6 is the hydrophilic and hydrophobic area of the middle layer, 7 is the upper layer liquid inlet, 8 is the upper layer liquid outlet, 9 upper liquid pool.

Detailed ways

The following examples will further illustrate the present invention, but do not limit the present invention thereby.

Example 1

Construction of Multilayer Paper Chips for Hepatocyte Solo Culture

The present invention is a multi-layer paper chip based on microfluidic technology and its construction method, as shown in Figure 1 as a whole. As shown in Figures 2a, 2b, 2c. The lower layer structure is composed of the lower layer liquid inlet 1, the lower layer liquid pool 3, and the lower layer liquid outlet 2, and the lower layer liquid pool 3 is communicated with the lower layer liquid inlet 1 and the lower layer liquid outlet 2;

The middle layer structure is composed of middle layer hole one 4, middle layer hole two 5 and middle layer hydrophilic and hydrophobic area 6, and the middle layer hydrophilic and hydrophobic area 6 is not connected with the middle layer hole;

The upper layer structure is composed of the upper layer liquid inlet 7, the upper layer liquid pool 9, and the upper layer liquid outlet 8; the upper layer liquid pool (9) is communicated with the upper layer liquid inlet 7 and the upper layer liquid outlet 8;

The lower layer liquid inlet 1 is communicated with the middle layer hole 4, and the lower layer liquid inlet 2 is communicated with the middle layer hole 5;

The channel is a straight channel, the channel is a single channel,

The hydrophilic and hydrophobic regions 6 of the intermediate layer are formed by a single circle.

The multi-layer paper chip based on microfluidic technology is formed by irreversible sealing of upper, middle and lower layers, the material of the upper layer and the lower layer are both light-transmitting and breathable PDMS polymers, and the material of the middle layer is photoresist patterned. Qualitative filter paper.

The SU8 glue (2025) patterned on the filter paper in the middle layer of the paper chip was diluted with cyclopentanone, and the volume ratio of SU8 glue to cyclopentanone was 1:1.

The middle layer filter paper of the paper chip has obvious hydrophilic and hydrophobic regions after patterning, and the upper and lower layers and both sides of the middle layer paper are plasma treated for 1.5 minutes and then sealed.

The present invention provides a method for constructing a multi-layer paper chip based on microfluidic technology. The basic process is as follows: (1) Patterned qualitative filter paper

After modifying the glass slide with trimethylchlorosilane vapor, bake it at 65°C for a while, then throw a layer of PDMS, heat to solidify the PDMS, put a certain size of qualitative filter paper on the PDMS, and use a syringe to drop a certain dilution ratio (1) on the paper. :1) SU8 glue (2025) to make it flat and the filter paper is saturated with SU8 glue, defoamed in a vacuum drying oven, pre-baked at 65 ℃, squeezed with a glass sheet with PDMS layer, naturally cooled, exposed, and then Bake, develop after natural cooling, blow-dry, and dry at 65°C for 5 minutes, to obtain a patterned paper in the middle layer, which is ready for use;

The trimethylchlorosilane steam modification time is 5min, the thickness of the PDMS on the glass sheet is 100 μm, the amount of SU8 glue dropped on the qualitative filter paper is 0.8mL, and the defoaming temperature range of the vacuum drying oven is 45°C;

The pattern on the filter paper is shown in Figure 2b, the structure formed by circular intervals, the pore size of the hydrophilic area is 400 μm, the hole interval is 200 μm, and the development time is 30 minutes; the structure of the intermediate layer paper prepared by the above method is characterized by scanning electron microscopy, and the results are shown in Figure 3 shown.

(2) Integrated microfluidic channel

The glass sheet was dewatered for 0.5h, and the surface of the glass was purified by oxygen for 1.5min. A certain thickness of SU8 glue was thrown, pre-baking, natural cooling, and partial exposure, post-baking, developing, and film hardening at 160 °C. After natural cooling, the template of SU8 was obtained. In order to make it hydrophobic, it was modified with trimethylchlorosilane, then reversed with PDMS, and cured at 65 °C to obtain a PDMS chip with a microfluidic channel structure. The upper and lower PDMS with the structure were treated with oxygen, etc. Both sides of the intermediate layer patterned paper prepared in step (1) are also treated with oxygen, etc., and then the upper and lower layers of PDMS with channels are irreversibly sealed with the intermediate layer patterned paper. The paper chip is flat and well sealed, that is, a multi-layer paper chip with integrated microfluidic channel is prepared, and the preparation process is shown in Figure 4;

The thickness of the SU8 glue is 150 μm, and the PDMS structure containing the microfluidic channel is a single straight channel. As shown in Figures 2a and 2c, the PDMS microfluidic liquid pools (3) and (9) channels are 10 mm long and 3 mm wide;

The oxygen treatment time for the upper and lower PDMS layers with the structure is 0.5 min, and the double-sided oxygen treatment time for the paper patterned in the middle layer is 1.5 min. Figure 5 shows the physical picture of the multilayer paper chip based on the microfluidic technology prepared according to the above steps.

This new multi-layer paper chip based on microfluidic technology has the following advantages over traditional two-dimensional paper chips: it can realize the co-culture of two kinds of cells, inoculate different cells in the upper liquid pool and the lower liquid pool, and explore the interaction between cells. Function; the paper chip with integrated microfluidic channel is a perfusable system, that is, the liquid can be perfused from the lower liquid inlet and the upper liquid inlet, thereby simulating the microenvironment and providing more favorable nutrients for cell growth; the construction of three-dimensional effects is a barrier study By building a technology platform, cells can be loaded on both sides of the intermediate paper chip to form a barrier, and nanoparticles and drugs can be added from the entrance of the lower or upper layer to further study the mechanism of its action on the barrier, etc. These characteristics are all traditional two-dimensional paper chips. The chip does not have it.

Hepatocytes were individually cultured using the above-prepared chip. It can also be used for other types of cells to be cultured alone, without limiting the present invention. Here, HepG2 was selected for hepatocytes, and HepG2 cells (cell density 4×10 ⁵ ) were inoculated at the upper liquid inlet (7), and the cells were individually cultured, which will not be described in detail here.

Example 2

Construction of multi-layer paper chips for co-culture of liver and islet cells

The present invention is a method for constructing a multi-layer paper chip based on microfluidic technology, as shown in Fig. 1 as a whole. 2a, 2b, and 2c. The lower layer structure is composed of the lower layer liquid inlet 1, the lower layer liquid pool 3, and the lower layer liquid outlet 2, and the lower layer liquid pool 3 is communicated with the lower layer liquid inlet 1 and the lower layer liquid outlet 2;

The middle layer structure is composed of two middle layer holes 4, 5 and the middle layer hydrophilic and hydrophobic area 6, and the middle layer hydrophilic and hydrophobic area 6 is not connected with the middle layer holes 4, 5;

The upper layer structure is composed of the upper layer liquid inlet 7, the upper layer liquid pool 9, and the upper layer liquid outlet 8; the upper layer liquid pool 9 is communicated with the upper layer liquid inlet 7 and the upper layer liquid outlet 8;

The lower layer liquid inlet 1 is communicated with the middle layer hole 4, and the lower layer liquid inlet 2 is communicated with the middle layer hole 5;

The channel is a straight channel, the channel is a single channel,

The hydrophilic and hydrophobic regions 6 of the intermediate layer are formed by a single circle.

The multi-layer paper chip based on microfluidic technology is formed by irreversible sealing of upper, middle and lower layers, the material of the upper layer and the lower layer are both light-transmitting and breathable PDMS polymers, and the material of the middle layer is photoresist patterned. Qualitative filter paper.

The SU8 glue (2025) patterned on the filter paper in the middle layer of the paper chip was diluted with cyclopentanone, and the volume ratio of SU8 glue to cyclopentanone was 1:3.

The middle layer filter paper of the paper chip has obvious hydrophilic and hydrophobic regions after patterning, and the upper and lower layers and both sides of the middle layer paper are plasma treated for 1.0 min and then sealed.

The invention provides a multi-layer paper chip based on microfluidic technology and a construction method thereof. The basic process is as follows:

(1) Patterned qualitative filter paper

After modifying the glass slide with trimethylchlorosilane vapor, bake it at 95° for a while, then throw a layer of PDMS, heat to solidify the PDMS, put a certain specification of qualitative filter paper on the PDMS, and use a syringe to drop a certain dilution ratio (1) on the paper. :3) SU8 glue (2025) to make it flat and the filter paper is soaked with SU8 glue, defoamed in a vacuum drying oven, pre-baked at 95 ℃, squeezed with a glass sheet with PDMS layer, naturally cooled, exposed, and then Bake, develop after natural cooling, blow-dry, and dry at 95°C for 10 minutes, to obtain a patterned paper in the middle layer, which is ready for use;

The trimethylchlorosilane steam modification time is 5min, the thickness of the PDMS on the glass sheet is 80 μm, the amount of SU8 glue dropped on the qualitative filter paper is 0.8mL, and the defoaming temperature range of the vacuum drying oven is 45°C;

The pattern on the filter paper is shown in Figure 2b, the structure formed by circular intervals, the pore diameter of the hydrophilic area is 400 μm, the hole interval is 200 μm, and the development time is 30 minutes; the structure of the intermediate layer paper prepared by the above method is characterized by scanning electron microscopy, and the results are shown in Figure 6 shown. It can be seen that the outline of the SU8 patterned paper diluted with cyclopentanone is clearer and the effect is better.

(2) Integrated microfluidic channel

The glass sheet was dewatered for 2 hours, and the surface of the glass was purified by oxygen for 1 minute. A certain thickness of SU8 glue was thrown, pre-baking, natural cooling, and partial exposure, post-baking, developing, and film hardening at 120 °C. After natural cooling, the template of SU8 was obtained. It is hydrophobic, so it is modified with trimethylchlorosilane, and then reversed with PDMS, and cured at 95 °C to obtain a PDMS chip with a microfluidic channel structure, and the upper and lower PDMS with the structure is treated with oxygen. The two sides of the interlayer patterned paper prepared in 1) were also treated with oxygen, etc., and then the upper and lower layers of PDMS with channels were irreversibly sealed with the interlayer patterned paper, and the integrated paper was extruded with a copper plate at 95 °C. The chip is flat and well sealed, that is, a multi-layer paper chip with integrated microfluidic channel is prepared, and the preparation process is shown in Figure 4;

The thickness of the SU8 glue is 200 μm, and the PDMS structure containing the microfluidic channel is a single straight channel. As shown in Figures 2a and 2c, the PDMS microfluidic liquid pools (3) and (9) channels are 10 mm long and 3 μm wide;

The oxygen treatment time of the upper and lower PDMS layers with the structure is 0.5 min, and the double-sided oxygen treatment time of the paper patterned in the middle layer is 1.0 min. Figure 5 shows the physical picture of the multilayer paper chip based on the microfluidic technology prepared according to the above steps.

Co-culture of liver and pancreas cells was carried out using the chip prepared above. It can also be used for co-cultivation of other kinds of cells without limiting the present invention. Here, HepG2 was selected for hepatocytes, and β-TC6 cells ^were selected for islet cells. ⁶ ), to carry out cell co-culture, which will not be described in detail here.

Example 3

Construction of multi-layer paper chips for islet cell culture alone

The present invention is a multi-layer paper chip based on microfluidic technology and its construction method, as shown in Figure 7 as a whole. As shown in Figures 8a, 8b, 8c. The lower layer structure is composed of the lower layer liquid inlet 1, the lower layer liquid pool 3, and the lower layer liquid outlet 2, and the lower layer liquid pool 3 is communicated with the lower layer liquid inlet 1 and the lower layer liquid outlet 2;

The middle layer structure is composed of middle layer hole one 4, middle layer hole two 5 and middle layer hydrophilic and hydrophobic area 6, and the middle layer hydrophilic and hydrophobic area 6 is not connected with the middle layer hole;

The upper layer structure is composed of the upper layer liquid inlet 7, the upper layer liquid pool 9, and the upper layer liquid outlet 8; the upper layer liquid pool 9 is communicated with the upper layer liquid inlet 7 and the upper layer liquid outlet 8;

The lower layer liquid inlet 1 is communicated with the middle layer hole 4, and the lower layer liquid inlet 2 is communicated with the middle layer hole 5;

The channel is straight, the channel is a single channel,

The hydrophilic and hydrophobic regions 6 of the intermediate layer are composed of straight channels in an array.

The multi-layer paper chip based on microfluidic technology is formed by irreversible sealing of upper, middle and lower layers, the material of the upper layer and the lower layer are both light-transmitting and breathable PDMS polymers, and the material of the middle layer is photoresist patterned. Qualitative filter paper.

The SU8 glue (2025) patterned on the middle layer filter paper of the paper chip was diluted with cyclopentanone, and the volume ratio of SU8 glue to cyclopentanone was 1:5.

The middle layer filter paper of the paper chip has obvious hydrophilic and hydrophobic regions after patterning, and the upper and lower layers and both sides of the middle layer paper are plasma treated for 2.0 minutes and then sealed.

The invention provides a multi-layer paper chip construction method based on microfluidic technology, and the basic process is as follows:

(1) Patterned qualitative filter paper

After modifying the glass piece with trimethylchlorosilane vapor, bake it at 80° for a while, then throw a layer of PDMS, heat to solidify the PDMS, put a certain size of qualitative filter paper on the PDMS, and use a syringe to drop a certain dilution ratio (1) on the paper. :5) SU8 glue (2025) to make it flat and the filter paper is saturated with SU8 glue, defoamed in a vacuum drying oven, pre-baked at 80 ℃, squeezed with a glass sheet with PDMS layer, naturally cooled, exposed, and then Bake, develop after natural cooling, blow-dry, and dry at 80°C for 15 minutes, to obtain a patterned paper in the middle layer, which is ready for use;

The trimethylchlorosilane steam modification time is 5min, the thickness of the PDMS on the glass sheet is 200 μm, the amount of SU8 glue dropped on the qualitative filter paper is 1.3mL, and the defoaming temperature range of the vacuum drying oven is 65°C;

The pattern on the filter paper is shown in Figure 8b, the structure formed by the array of straight channels, the straight channel is 6 mm long, 300 μm wide, 300 μm in spacing, and the development time is 20 min.

(2) Integrated microfluidic channel

The glass sheet was dewatered for 1.5 hours, and the surface of the glass was purified by oxygen for 2 minutes. A certain thickness of SU8 glue was thrown, pre-baking, natural cooling, and partial exposure, post-baking, developing, and film hardening at 180 °C. After natural cooling, the template of SU8 was obtained, which is To make it hydrophobic, it was modified with trimethylchlorosilane, and then reversed with PDMS, and cured at 80 °C to obtain a PDMS chip with a microfluidic channel structure. The upper and lower PDMS with the structure were treated with oxygen. Both sides of the interlayer patterned paper prepared in (1) were also treated with oxygen, etc., and then the upper and lower PDMS layers with channels were irreversibly sealed with the interlayer patterned paper, and the integrated The paper chip is flat and well sealed, that is, a multi-layer paper chip with integrated microfluidic channel is prepared, and the preparation process is the same as that shown in Figure 4;

The thickness of the SU8 glue is 500 μm, and the PDMS structure containing the microfluidic channel is a single straight channel. As shown in Figures 2a and 2c, the PDMS microfluidic liquid pools (3) and (9) channels are 10 mm long and 3 mm wide;

The oxygen treatment time of the upper and lower PDMS layers with the structure is 0.5 min, and the double-sided oxygen treatment time of the paper patterned in the middle layer is 2.0 min.

The above-prepared chip is used to culture islet cells alone, and islet cells (cell density 2×10 ⁷ ) are inoculated at the upper liquid inlet (7) for individual cell culture, which will not be described in detail here.

Claims (9)

1. a multi-layer paper chip based on microfluidic technology, is characterized in that: this paper chip is mainly formed by three-layer structure sealing, including lower layer structure, middle layer structure and upper layer structure; The lower layer structure is composed of a lower layer liquid inlet (1), a lower layer liquid pool (3), and a lower layer liquid outlet (2), and the lower layer liquid pool (3) is communicated with the lower layer liquid inlet (1) and the lower layer liquid outlet (2); The middle layer structure is composed of middle layer hole one (4), middle layer hole two (5) and middle layer hydrophilic and hydrophobic area (6), and the middle layer hydrophilic and hydrophobic area (6) is not connected with the middle layer hole; The upper layer structure is composed of the upper layer liquid inlet (7), the upper layer liquid pool (9), and the upper layer liquid outlet (8); the upper layer liquid pool (9) is communicated with the upper layer liquid inlet (7) and the upper layer liquid outlet (8); The lower layer liquid inlet (1) is communicated with the middle layer hole (4), and the lower layer liquid inlet (2) is communicated with the middle layer hole (5); the middle layer hydrophilic and hydrophobic zone (6), the upper layer liquid pool (3), the lower layer liquid pool ( 9) Connected by channel, The channel is a straight channel or a curved channel, The channel is single channel or multi-channel, The hydrophilic and hydrophobic regions (6) of the intermediate layer are composed of a single circle or a plurality of patterns. 2. A kind of multi-layer paper chip based on microfluidic technology according to claim 1, it is characterized in that: described paper chip is formed by irreversible sealing of upper, middle and lower layers, and the material of upper layer and lower layer are both light-transmitting Breathable PDMS polymer, the intermediate layer material is photoresist patterned qualitative filter paper. 3. a kind of multi-layer paper chip based on microfluidic technology according to claim 2, is characterized in that: the intermediate layer material of described paper chip, qualitative filter paper has obvious affinity and phobia by patterning of SU8 glue of certain dilution ratio water area. 4. a kind of multilayer paper chip based on microfluidic technology according to claim 3, is characterized in that: the SU8 glue of patterning is diluted with cyclopentanone, and SU8 glue and cyclopentanone volume ratio are 1:1- 5. 5. the construction method of a kind of multilayer paper chip based on microfluidic technology according to claim 1 is characterized in that carrying out according to the following steps: (1) Patterned qualitative filter paper After modifying the glass piece with trimethylchlorosilane vapor, bake it at 50℃-95℃ for a while, then throw a layer of PDMS, heat to solidify the PDMS, put a certain specification of qualitative filter paper on the PDMS, and use a syringe to drop a certain amount of dilution on the paper Multiple times of SU8 glue to make it flat and the filter paper is saturated with SU8 glue, defoaming in a vacuum drying oven, pre-baking at 60 ℃-95 ℃, extruding with a glass sheet with PDMS layer, natural cooling, exposure, post-baking, After natural cooling, develop, blow-dry, and dry at 60°C-95°C for 5-30min, that is, the paper with patterned middle layer is prepared, which is ready for use; (2) Integrated microfluidic channel Dewater the glass sheet for 0.5-2h, oxygen and other surface purification treatment for 1-3min, throw a certain thickness of SU8 glue, pre-bake, natural cooling, and then partially expose, post-bake, develop, harden the film at 90℃-180℃, and naturally cool the back side The template of SU8 was obtained. In order to make it hydrophobic, it was modified with trimethylchlorosilane, and then reversed with PDMS. After curing at 60°C-95°C, a PDMS chip containing a microfluidic channel structure can be obtained. Treated with oxygen, etc. Both sides of the upper and lower layer PDMS with structure and the interlayer patterned paper prepared in step (1), and then irreversibly sealing the upper and lower layer PDMS with channels and the interlayer patterned paper, at 60°C-95°C The integrated paper chip is made flat and well-sealed by extrusion with a copper plate, that is, a multi-layer paper chip with integrated microfluidic channel is prepared. 6. the construction method of the multi-layer paper chip based on microfluidic technology according to claim 5, is characterized in that in step (1): trimethyl chlorosilane steam modification time is 1-10min, the thickness of PDMS is thrown on the glass sheet It is 50-200μm, the amount of SU8 glue with a certain dilution ratio dropped on the qualitative filter paper is 0.6-1.3mL, and the defoaming temperature range of the vacuum drying box is 40℃-70℃. 7. The method for constructing a multi-layer paper chip based on microfluidic technology according to claim 5, wherein in step (1): the pattern on the patterned paper of the intermediate layer is a structure formed by circular intervals, Straight channel structure, serpentine structure or other structures, the development time is 20-50min. 8. the construction method of the multi-layer paper chip based on microfluidic technology according to claim 5, is characterized in that in step (2): SU8 glue thickness is 50-500 μm, and described PDMS chip containing microfluidic channel, The channel is a single straight channel, a serpentine channel, a plurality of parallel channels or channels of other structures. 9. the construction method of a kind of multi-layer paper chip based on microfluidic technology according to claim 5, is characterized in that in step (2): oxygen etc. process the upper and lower PDMS time with structure is 0.5-3min, the middle The double-sided oxygen treatment time of the layer-patterned paper is 1-5 min.

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