Preparation method of sandwich structure microfluidic chip and fluid filling method

Abstract

The present invention discloses a method for preparing a microfluidic chip with a sandwich structure using a flexible mold peeling process, using a flexible substrate and a patterned photoresist as a flexible mold, forming a flexible thin layer on the flexible mold, bonding the composite structure of the flexible thin layer and the flexible mold to a hard bottom layer, and obtaining a flow path structure corresponding to the photoresist pattern on the flexible thin layer after peeling the flexible mold, and then bonding a hard cover plate on the surface of the flexible thin layer flow path structure to achieve a low-cost and high-yield sandwich structure microfluidic chip, and the middle layer is ultra-thin and flexible. In addition, for a microfluidic chip with a sandwich structure having an intermediate layer of a breathable material prepared by a flexible mold peeling process, a fluid filling method is proposed to form multiple independent reaction units, and the liquid evaporation amount of the microfluidic chip is small, which can be applied in the fields of protein crystallization and digital PCR that require reaction heating.

Description

Technical Field

[0001] The present application relates to the technical field of microfluidic chips and related applications, and in particular to a method for preparing a microfluidic chip with a sandwich structure using a flexible mold peeling process and a fluid filling method. Background Art

[0002] Polydimethylsiloxane (PDMS) is a widely used basic material in the field of microfluidic chips. However, due to the small molecule diffusion and gas permeability of PDMS material, which causes the volatilization of reagents, it is difficult to effectively carry out reactions that require a long time, such as protein crystallization, digital PCR, and cell culture.

[0003] Now there is a solution to add glass barriers on both sides of the thin PDMS flow channel to form a glass-PDMS-glass sandwich structure microfluidic chip to reduce the evaporation of reagents in the microfluidic chip during the reaction process. Among them, the preparation of the thin PDMS layer is more critical. There are methods of directly spin-coating a thin layer of PDMS, PET or high-temperature tape to transfer about 100um thick PDMS to prepare glass-PDMS-glass microfluidic chips. Compared with PDMS chips, the amount of reagent evaporation is smaller, but the yield rate is lower. Summary of the invention

[0004] In view of the problems and shortcomings of the existing technical solutions, the present invention discloses a method for preparing a microfluidic chip with a sandwich structure using a flexible mold peeling process, which solves the problem that the existing glass-PDMS-glass microfluidic chip technical solution cannot achieve low-cost and high-yield production of ultra-thin flexible film sandwich structure microfluidic chips. The following technical solutions are adopted:

[0005] A flexible substrate and a patterned photoresist are used as a flexible mold, a flexible thin layer is formed on the flexible mold, and the composite structure of the flexible thin layer and the flexible mold is bonded to a hard base layer. After the flexible mold is peeled off, a flow path structure corresponding to the photoresist pattern is obtained on the flexible thin layer, and then a glass cover plate is bonded to the surface of the flexible thin layer flow path structure to realize a low-cost and high-yield sandwich-structured microfluidic chip, and the middle flexible thin layer is ultra-thin.

[0006] The present invention provides a method for preparing a microfluidic chip with a sandwich structure, comprising the following steps:

[0007] 1) Attaching the flexible substrate to a rigid base;

[0008] 2) coating a photoresist on the surface of the flexible substrate and patterning the photoresist;

[0009] 3) coating a flexible thin layer on the patterned photoresist, wherein the thickness of the flexible thin layer is 20 μm-500 μm;

[0010] 4) separating the composite structure of the flexible substrate, the photoresist and the flexible thin layer from the rigid base;

[0011] 5) bonding the flexible thin layer surface of the composite structure to the hard bottom layer;

[0012] 6) peeling the flexible substrate and the photoresist from the flexible thin layer, and obtaining a flow path structure corresponding to the photoresist pattern on the surface of the flexible thin layer;

[0013] 7) Bonding the hard cover plate to the patterned surface of the flexible thin layer.

[0014] Optionally, the flexible substrate includes a flexible polyester (PET) film, a flexible polymethyl methacrylate (PMMA) film, a flexible polycarbonate film (PC) film, a flexible cycloolefin copolymer (COC) film, a flexible polyvinyl chloride (PVC) film or a high-temperature tape, with a thickness of 100 μm-1000 μm.

[0015] Optionally, the photoresist has a thickness of 5 μm-80 μm.

[0016] Optionally, the flexible thin layer includes polydimethylsiloxane (PDMS), polyvinyl alcohol (PVA), polyester (PET), polyimide (PI), polyethylene naphthalate (PEN), paper, textile material, etc.

[0017] Optionally, the flow path structure is distributed in a flexible thin layer.

[0018] Optionally, the hard cover plate is provided with an inlet hole and an outlet hole, the inlet hole is connected to the flow path in the flexible thin layer, and the hard cover plate and the hard bottom layer include materials such as glass, silicon, polymethyl methacrylate (PMMA), etc.

[0019] Optionally, the bonding method includes plasma bonding, mechanical clamping bonding or thermal bonding.

[0020] Optionally, in the step 2), the surface of the flexible substrate is firstly subjected to plasma treatment to obtain a surface having surface activity, and then a positive photoresist is coated on the surface having surface activity.

[0021] Optionally, the thickness of the flexible thin layer is 20 μm-200 μm.

[0022] Optionally, the thickness of the hard cover plate and the hard bottom layer is 500 μm-3 mm respectively.

[0023] A microfluidic chip with a sandwich structure of a hard bottom layer, a flexible thin layer and a hard cover plate is prepared by the above preparation method. The present invention provides a method for filling a fluid in a microfluidic chip with a sandwich structure, comprising the following steps:

[0024] Step 1, fixing and clamping a sandwich structure microfluidic chip consisting of a hard bottom layer, a flexible thin layer, and a hard cover plate, and injecting fluid 1 into it through a liquid inlet pipe, and after flowing through the flow channel of the microfluidic chip, flowing to the outlet hole, some liquid overflows;

[0025] Step 2, sealing the outlet hole, and continuously passing fluid 1 into the microfluidic chip, so that fluid 1 fills the entire flow channel of the microfluidic chip;

[0026] Step 3, the outlet hole is no longer sealed, and fluid 2 is injected into the microfluidic chip. After passing through part of the flow channels of the microfluidic chip, fluid 2 flows to the outlet hole and overflows part of the liquid. At this time, fluid 1 is retained in the flow channels of the microfluidic chip that do not flow through fluid 2, and multiple independent reaction units can be formed;

[0027] Step 4, after sealing the inlet hole and the outlet hole of the microfluidic chip, the microfluidic chip can be heated and other operations can be performed, wherein the evaporation amount of the liquid is less than that of the microfluidic chip without a hard cover.

[0028] Necessarily, the flexible thin layer is made of PDMS or other materials with certain air permeability;

[0029] Optionally, the method of injecting fluid into the chip can be to use an air pressure / syringe pump to form a positive pressure at the liquid inlet to push the liquid, and a syringe pump / vacuum box to form a negative pressure in the chip flow channel at the liquid outlet, and then suck the liquid dripped at the liquid inlet into the flow channel;

[0030] Optionally, the sealing method may be to plug the corresponding orifice with a steel needle, tape, plug, etc.;

[0031] The beneficial effects of the present invention are:

[0032] (1) The sandwich structure microfluidic chip prepared by the flexible mold peeling process can realize the manufacture of the sandwich structure microfluidic chip with a minimum thickness of 20 μm of flexible thin layer, with low cost and high yield;

[0033] (2) For a microfluidic chip with a sandwich structure and a PDMS middle layer prepared by a flexible mold peeling process, a fluid filling method is proposed to form multiple independent reaction units. The liquid evaporation of the microfluidic chip is small, and it can be applied in fields such as protein crystallization and digital PCR that require reaction heating. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Schematic diagram of the structure of the microfluidic chip with a micro-chamber digital PCR sandwich structure of the embodiment, wherein (a) is a three-dimensional structure diagram, and (b) is a top view of the PDMS thin layer;

[0035] Figure 2-12 This is a manufacturing flow chart of a glass-ultra-thin PDMS-glass sandwich structure microfluidic chip using a flexible mold peeling process in an embodiment, and the structure obtained in each step is shown in the figure;

[0036] Figure 13-16 Schematic diagram of the flow of a fluid filling method for a sandwich structure microfluidic chip of an embodiment;

[0037] Fig.17 Comparison of microscope images after evaporation of glass-PDMS-glass micro-chamber digital PCR microfluidic chip. The scales of each image are consistent, and the black part is dye; microscope image before thermal cycling (a1) PDMS thickness 5mm, (a2) PDMS thickness 200μm; microscope image after thermal cycling at 95℃, 10min (b1) PDMS thickness 5mm, (b2) PDMS thickness 200μm; microscope image after 45 thermal cycles (65℃, 30s & 95℃, 30s) (c1) PDMS thickness 5mm, (c2) PDMS thickness 200μm;

[0038] Description of reference numerals:

[0039] 100, glass bottom layer; 200, PDMS thin layer; 201, fluid dispersion channel; 202, arrayed circular cavity; 203, channel connecting each chamber; 204, fluid convergence channel; 210, first surface; 220, second surface; 300, glass cover; 301, inlet hole; 302, outlet hole; 400, silicon wafer; 410, polishing surface; 420, lower surface; 500, flexible PET film; 510, bare PET surface; 520, adhesion surface; 530, surface-active PET surface; 600, AZ-4620 photoresist; 610, patterned photoresist; 700, microfluidic chip fixing fixture; 801, liquid inlet pipe; 802, liquid outlet pipe; 803, steel needle; 910, fluid one; 920, fluid two. DETAILED DESCRIPTION

[0040] The present invention is further explained below in conjunction with the accompanying drawings and specific embodiments. The various drawings of the present invention are only for illustration to make it easier to understand the present invention, and the specific proportions can be adjusted according to design requirements. The upper and lower relationships of the relative elements and the definitions of the front / back in the figures described in the text should be understood by those skilled in the art to refer to the relative positions of the components, so they can all be flipped to present the same components, which should all fall within the scope disclosed in this specification.

[0041] The following is combined with Figure 1-12 This application is described in further detail.

[0042] Figure 1It is a structural schematic diagram of a micro-chamber digital PCR sandwich structure microfluidic chip, which includes a glass bottom layer (hard bottom layer) 100, a PDMS thin layer (flexible thin layer) 200 and a glass cover plate (hard cover plate) 300.

[0043] The flow path structure in the PDMS layer 200 includes a fluid dispersion channel 201, an array of circular cavities 202, a channel 203 connecting each chamber, and a fluid convergence channel 204, and the glass cover plate 300 has an inlet hole 301 and an outlet hole 302. The PDMS layer 200 is sandwiched between the glass bottom layer 100 and the glass cover plate 300, and the fluid enters the fluid dispersion channel 201 from the inlet hole 301, enters the array of circular cavities 202 through the channel 203 connecting each chamber, and flows out from the outlet hole 302 through the fluid convergence channel 204, forming a flow path.

[0044] A method for preparing a microfluidic chip with a sandwich structure using a flexible mold peeling process comprises the following steps:

[0045] Step 1, such as Figure 2 As shown, a silicon wafer 400 with a polished surface 410 is selected as a rigid substrate and a flexible PET film 500 is selected as a flexible substrate. The flexible PET film 500 includes a bare PET surface 510 and an adhesive surface 520. The thickness of the flexible PET film is 500 um.

[0046] Step 2, such as Figure 3 As shown, the adhesive surface 520 of the flexible PET film 500 is attached to the polished surface 410 of the silicon wafer 400 to ensure the flatness of the flexible PET film 500 and that there are no bubbles between the polished surface 410 and the adhesive surface 520;

[0047] Step 3, such as Figure 4 As shown, the PET surface 510 of the flexible PET film 500 is subjected to plasma treatment to obtain a PET surface 530 with surface activity, which can enhance the adhesion between the flexible PET film 500 and the AZ-4620 photoresist 600 in step 4;

[0048] Step 4, such as Figure 5 As shown, the surface active PET surface 530 in step 3 is spin coated with AZ-4620 photoresist 600 once or multiple times;

[0049] Preferably, the thickness of the spin-coated AZ-4620 photoresist 600 is 10-50 um;

[0050] Step 5, such as Figure 6As shown, the AZ-4620 photoresist 600 spin-coated in step 4 is baked, photolithographically processed, developed and hardened to obtain a patterned photoresist 610; the pattern of the patterned photoresist 610 corresponds to the flow path structure on the preset PDMS layer, and the thickness of the photoresist is the depth of the flow channel.

[0051] Step 6, such as Figure 7 As shown, the lower surface 420 of the silicon wafer 400 obtained in step 5 is adsorbed on a coating machine, and a PDMS thin layer 200 is spin-coated on the patterned photoresist 610, heated to cure and cooled;

[0052] Preferably, the PDMS thin layer uses a polymer and a cross-linking agent in a weight ratio of 10:1;

[0053] Preferably, the PDMS thin layer 200 is 200 um thick and the heating curing temperature is 120°C.

[0054] Step 7, such as Figure 8 As shown, the flexible PET film 500, the patterned photoresist 610 and the PDMS thin layer 200 are peeled off from the silicon wafer 400 together;

[0055] Preferably, the flexible PET film 500, the patterned photoresist 610 and the PDMS thin layer 200 are flexible and can be easily peeled off from the hard silicon wafer 400, and the deformation caused to the fluid dispersion channel 201, the arrayed circular cavity 202, the channel 203 connecting the chambers and the fluid convergence channel 204 in the PDMS thin layer 200 is almost negligible;

[0056] Step 8, such as Fig. 9 As shown, after the structure obtained in step 7 is flipped 180°, the first surface 210 of the PDMS thin layer 200 is bonded to the glass bottom layer 100 by oxygen plasma treatment;

[0057] Step 9, such as Figure 10-11 As shown in the figure, the adhesion between the patterned photoresist 610 and the flexible PET film 500 is greater than the bonding force between the patterned photoresist 610 and the PDMS thin layer 200, and the flexible mold composed of the flexible PET film 500 and the patterned photoresist 610 can be peeled off from the PDMS thin layer 200. The specific method is to clamp a corner of the flexible substrate with tweezers and slowly lift it up along the direction of the flow channel in the PDMS thin layer 200, so as to achieve non-destructive peeling of the PDMS, and then use acetone, alcohol and deionized water to clean it in turn;

[0058] Step 10, such as Fig.12 As shown, a glass cover plate 300 with an inlet hole 301 and an outlet hole 302 is bonded to the second surface 220 of the PDMS thin layer 200;

[0059] Preferably, the thickness of the glass bottom layer and the glass cover plate is 1 mm.

[0060] A microfluidic chip with a sandwich structure of a hard bottom layer, a flexible thin layer and a hard cover plate is prepared by the above preparation method. The present invention provides a method for filling a fluid in a microfluidic chip with a sandwich structure, comprising the following steps:

[0061] Step 1, such as Fig.13 As shown, a microfluidic chip fixing fixture 700 is used to fix and clamp a sandwich structure microfluidic chip consisting of a hard bottom layer 100, a flexible thin layer 200 and a hard cover plate 300, and a fluid 910 is injected into it through a liquid inlet pipe 801. After passing through the inlet hole 301 and the flow channel 203 connecting each chamber, it flows to the outlet hole 302 and then overflows part of the liquid through the liquid outlet pipe 802;

[0062] Step 2, such as Fig.14 As shown, the outlet hole 302 is sealed by a steel needle 803, and the fluid 1 910 is continuously passed into the microfluidic chip, so that the fluid 1 910 fills the entire flow channel of the microfluidic chip including the array of circular cavities 202 and the flow channels 203 connecting the chambers, without bubbles;

[0063] Step 3, such as Fig.15 As shown, after the steel needle 803 is removed, fluid 2 920 is injected into it through the liquid inlet pipe 801, and after flowing through the inlet hole 301 and the flow channel 203 connecting the chambers, it flows to the outlet hole 302 and overflows part of the liquid through the liquid outlet pipe 802. At this time, the fluid 1 910 in the flow channel 203 connecting the chambers is pushed away, while the fluid 1 910 in the array circular cavity 202 is still retained, thereby forming a large number of independent liquid reaction units;

[0064] Preferably, the flexible thin layer is made of PDMS material with certain air permeability;

[0065] Preferably, the method of injecting the fluid into the chip can use an air pressure pump to form a positive pressure at the liquid inlet to push the liquid;

[0066] Preferably, fluid 1 910 and fluid 1 920 are incompatible with each other and do not react with each other;

[0067] Step 4, such as Fig.16 As shown, two steel needles 803 respectively seal the liquid inlet channel 801 and the liquid outlet channel 802 of the microfluidic chip, and then heat the microfluidic chip.

[0068] like Fig.17The glass-PDMS-glass micro-cavity digital PCR microfluidic chip with the middle PDMS thin layer thickness of 5 mm and 200 um was processed by the above steps. The dye (black) was introduced and the thermal cycle conditions were hot start (95°C, 10 min) and 45 thermal cycles (65°C, 30 s&95°C, 30 s). The 200 um glass-PDMS-glass micro-cavity digital PCR microfluidic chip had a large amount of dye (black) remaining and the evaporation amount was significantly reduced, which had better performance in inhibiting water evaporation.

[0069] The above embodiments are only used to further illustrate the preparation method and fluid filling method of a sandwich-structured microfluidic chip of the present invention, but the present invention is not limited to the embodiments. Any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention fall within the protection scope of the technical solution of the present invention.

Claims

1. A method for preparing a sandwich structure microfluidic chip, characterized in that: The following steps are involved: 1) attaching a flexible substrate to a rigid substrate; the flexible substrate includes a flexible polymethyl methacrylate film, a flexible polycarbonate film, a flexible cycloolefin copolymer film, a flexible polyvinyl chloride film or a high-temperature tape, with a thickness of 100 μm-1000 μm; 2) Plasma treatment is performed on the surface of the flexible substrate to obtain a surface with surface activity, and then a positive photoresist is coated on the surface with surface activity to pattern the photoresist; 3) coating a flexible thin layer on the patterned photoresist, wherein the thickness of the flexible thin layer is 20 μm-500 μm; the material of the flexible thin layer includes polydimethylsiloxane, polyvinyl alcohol, polyimide, polyethylene naphthalate, paper or textile material; the thickness of the flexible thin layer is 20 μm-200 μm; 4) separating the composite structure of the flexible substrate, the photoresist and the flexible thin layer from the rigid substrate; 5) Bonding the flexible thin layer surface of the composite structure to the hard bottom layer; 6) peeling the flexible substrate and the photoresist from the flexible thin layer, and obtaining a flow path structure corresponding to the photoresist pattern on the surface of the flexible thin layer; 7) Bonding the hard cover plate to the patterned surface of the flexible thin layer.

2. The method for preparing a sandwich structure microfluidic chip according to claim 1, characterized in that: The thickness of the photoresist is 5 μm-80 μm.

3. The method for preparing a sandwich structure microfluidic chip according to claim 1, characterized in that In step 5) and step 7), the bonding method includes plasma bonding, mechanical clamping bonding or thermal bonding.

4. The method for preparing a sandwich structure microfluidic chip according to claim 1, characterized in that: The hard cover plate is provided with an inlet hole and an outlet hole, and the inlet hole is connected to the flow path structure in the flexible thin layer; the materials of the hard cover plate and the hard bottom layer include glass, silicon or polymethyl methacrylate; the thickness of the hard cover plate and the hard bottom layer is 500μm-3mm respectively.

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