Preparation method of structured PDMS thin layer / hard substrate complex

Abstract

The invention relates to the technical field of micro-fluidic chips, and discloses a preparation method of a structured PDMS thin layer / hard substrate complex. The preparation method comprises the following steps: S1, coating the surface of a hard substrate with a PDMS prepolymer, and carrying out standing reflux to form a uniform PDMS thin layer; s2, carrying out pre-curing treatment on the PDMS thin layer to obtain a semi-cured PDMS thin layer; s3, pressing a mold on the surface of the semi-cured PDMS thin layer, and filling the mold microstructure with the semi-cured PDMS under the assistance of vacuum; and S4, heating and curing, and stripping the mold to obtain the structured PDMS thin layer / hard substrate complex. According to the preparation method provided by the invention, through the synergistic effect of the steps, the structured PDMS thin layer / hard substrate complex with high rigidity, low moisture volatilization, solvent swelling resistance and microscopic compatibility is obtained, and the application requirements of different scenes on PDMS microfluidic devices are met.

Description

Technical Field

[0001] The present invention relates to the technical field of microfluidic chips, and particularly relates to a method for preparing a structured PDMS thin layer / rigid substrate composite body. Background Art

[0002] Polydimethylsiloxane (PDMS) has been widely used in the processing and fabrication of microfluidic chips due to its low cost, easy molding, good biocompatibility, excellent optical transparency, and chemical inertness. At present, PDMS microfluidic chips are mainly formed by irreversibly or reversibly sealing and assembling a patterned PDMS sheet (usually with a thickness of 1-4 mm) with a planar substrate (usually glass or PDMS). The obtained all-PDMS and PDMS-glass hybrid microfluidic chips have been widely used in many fields.

[0003] However, the PDMS microfluidic chips prepared by the above method have the following obvious defects: ① Insufficient stiffness: Due to the low Young's modulus of the PDMS material, the PDMS microfluidic chips have insufficient stiffness, resulting in pipeline deformation under the action of fluid pressure and collapse of low aspect ratio structures; ② Severe water evaporation: Due to the strong water molecule permeability of the PDMS material, the PDMS microfluidic chips have severe water evaporation, which is not conducive to long-term experiments (such as cell culture, protein crystallization, etc.) and high-temperature experiments (such as PCR); ③ Compatibility problems: Due to the easy swelling of the PDMS material under the action of most organic solvents, there are compatibility problems between the PDMS microfluidic chips and many commonly used solvents for biomolecule detection; ④ Incompatible with high-resolution microscopic observation: Due to the extremely short physical working distance of high-magnification objective lenses, for example, the working distance of a 40× / 60× objective lens is about 0.2-0.3 mm, while the thickness of a conventional PDMS chip is greater than 1 mm, exceeding the effective focusing range of the objective lens, resulting in unclear imaging.

[0004] Preparing a structured PDMS film-glass composite structure is an effective way to solve the above defects. However, the traditional structured PDMS manufacturing process usually involves pouring a PDMS prepolymer onto a mold, heating and curing it, and then peeling the PDMS from the mold to obtain a structured PDMS sheet (usually with a thickness of 1-4 mm). This process requires peeling, transferring, and bonding the PDMS structure layer. Since the PDMS thin film structure layer must overcome the adhesive forces such as van der Waals forces and hydrogen bonds between the PDMS thin film and the mold during the peeling and transfer process, and the PDMS thin film itself has a small thickness and low mechanical strength, it is very easy to have tearing, wrinkling, and microstructural damage during the peeling process, and the operation is very difficult and the yield is low, resulting in great limitations in the application of this process in fabricating microfluidic devices with integrated PDMS thin film microstructures.

[0005] In the past few decades, researchers have proposed various methods for fabricating structured PDMS thin films. For example, a glass slide is directly pressed onto the PDMS prepolymer poured into a silicon wafer mold. After the PDMS is completely cured, a blade is used to assist in separating the rigid glass-PDMS composite from the mold. Since the mechanical strength of the glass-PDMS composite is better than that of the PDMS film, the PDMS film is not easily torn or wrinkled during peeling. However, due to the very strong adhesion between the glass-PDMS composite and the mold, the glass substrate is easily broken and the silicon-based mold is damaged during the separation process, resulting in a low process success rate. Another example is to spin-coat a PDMS thin film layer on the substrate surface and combine it with reactive ion etching (RIE) technology to replace the traditional casting and molding method, and use the method of directly patterning the PDMS film layer on the substrate to fabricate a structured PDMS thin film. Although this method omits the transfer and bonding steps, since each chip needs to be independently lithographed and RIE processed, and relies on expensive professional equipment and highly skilled operators, it is difficult to popularize in ordinary laboratories and has low practicability.

[0006] Therefore, there is an urgent need to develop a method for fabricating a structured PDMS thin film / rigid substrate composite with simple operation, high efficiency, strong reliability, and high yield, which is used to develop microfluidic devices integrated with structured PDMS thin films to meet different application requirements. Summary of the Invention

[0007] The present invention aims to at least solve one of the above technical problems existing in the prior art. For this reason, one of the purposes of the present invention is to provide a method for preparing a structured PDMS thin film / rigid substrate composite.

[0008] Another purpose of the present invention is to provide a structured PDMS thin film / rigid substrate composite.

[0009] Another purpose of the present invention is to provide the application of this structured PDMS thin film / rigid substrate composite.

[0010] In order to achieve the above purposes, the technical solutions adopted by the present invention are as follows:

[0011] The first aspect of the present invention provides a method for preparing a structured PDMS thin film / rigid substrate composite, including the following steps:

[0012] S1. Coat the PDMS prepolymer on the surface of the rigid substrate, let it stand and reflux to form a uniform PDMS thin film;

[0013] S2. Perform pre-curing treatment on the PDMS thin film to obtain a semi-cured PDMS thin film;

[0014] S3. Press the mold onto the surface of the semi-cured PDMS thin film, and use vacuum assistance to fill the microstructures of the mold with the semi-cured PDMS;

[0015] S4. Heat and cure, then peel off the mold to obtain the structured PDMS thin layer / hard substrate composite.

[0016] In some embodiments of the present invention, the hard substrate is selected from glass, silicon wafers, metal sheets or polymer substrates.

[0017] In some preferred embodiments of the present invention, the metal sheet is selected from aluminum sheets, copper sheets or stainless steel sheets.

[0018] In some preferred embodiments of the present invention, the metal sheet is polished before use.

[0019] In some preferred embodiments of the present invention, the polymer substrate is selected from polycarbonate (PC) substrates, polymethyl methacrylate (PMMA) substrates or polyethylene terephthalate (PET) substrates.

[0020] In some preferred embodiments of the present invention, the hard substrate is selected from glass or silicon wafers.

[0021] In some embodiments of the present invention, the surface of the hard substrate is pretreated with oxygen plasma before use.

[0022] Specifically, in the present invention, glass, silicon wafers, metal sheets or polymer substrates are used as hard substrates. Such substrates have high stiffness. After the PDMS layer is combined with them, by transferring the load to the rigid substrate, stress and strain can be reduced. By using interfacial bonding to fix the geometry of PDMS, local instability can be prevented, thereby effectively suppressing the deformation of the PDMS layer under fluid pressure. It is preferred to use glass or silicon wafers as hard substrates. While providing high stiffness, the high surface flatness is more conducive to the uniform formation of the PDMS thin layer.

[0023] In some embodiments of the present invention, in step S1, in the PDMS prepolymer, the mass ratio of the silicon-based polymer to the curing agent is (5 - 20):1.

[0024] In some preferred embodiments of the present invention, in the PDMS prepolymer, the mass ratio of the silicon-based polymer to the curing agent is (5 - 15):1.

[0025] In some embodiments of the present invention, the PDMS prepolymer includes Dow Corning Sylgard 184 PDMS.

[0026] Specifically, in the PDMS prepolymer selected in the present invention, the silicone polymer itself is a linear or branched polymer. Triggered by a curing agent, a cross-linking reaction can occur to form a three-dimensional network structure, realizing the transformation from a liquid state to an elastomer. This process is a key step for the PDMS material to obtain functionality (elasticity, stability, etc.). When the mass ratio of the silicone polymer to the curing agent is too small (excessive curing agent), the cross-linking will be too fast, the material will become brittle, and it is easy to generate bubbles or internal stress cracks. When the mass ratio is too large (insufficient curing agent), the cross-linking will be incomplete, the PDMS will be too soft, and its anti-deformation ability will be poor. The PDMS prepolymer with the mass ratio of the silicone polymer to the curing agent within a specific range selected in the present invention has a precisely regulated cross-linking density and reaction rate, and the curing rate is balanced with the mechanical properties, which is more conducive to the preparation of a structured PDMS thin layer.

[0027] In some embodiments of the present invention, in step S1, the time for static reflux is 10 - 20 min.

[0028] In some preferred embodiments of the present invention, in step S1, the time for static reflux is 13 - 17 min.

[0029] Specifically, if the static reflux time is too short, the PDMS prepolymer will not be fully leveled, resulting in uneven thickness and distorted microstructure replication. If the static reflux time is too long, the viscosity of the prepolymer will increase, affecting the subsequent mold pressing and filling effect. By controlling the static reflux time to be 10 - 20 min (optimally 15 min) in the present invention, edge accumulation is eliminated, a uniform thin layer is formed, and accurate replication of the subsequent structure is ensured.

[0030] In some embodiments of the present invention, in step S2, the conditions for pre-curing treatment are selected from any one of the following:

[0031] a) 70 - 75°C, heating for 15 - 20 min;

[0032] b) 80 - 85°C, heating for 10 - 15 min;

[0033] c) 90 - 100°C, heating for 5 - 10 min;

[0034] d) Room temperature, standing for 10 - 12 h.

[0035] In some preferred embodiments of the present invention, in step S2, the conditions for pre-curing treatment are 70 - 75°C, heating for 15 - 20 min.

[0036] Specifically, in the present invention, the PDMS thin layer is first pre-cured. The obtained semi-cured PDMS material has a certain viscosity and fluidity but is not fully cross-linked, which is convenient for the filling of microstructures during mold pressing. At the same time, most of the polymer molecules in the semi-cured PDMS thin layer have been cross-linked, avoiding the formation of strong adhesion between the PDMS polymer molecules and the mold surface during the mold pressing and forming process, and reducing the difficulty of demolding after forming. During the semi-curing process, if the heating temperature is too high or the time is too long, it will cause excessive cross-linking of PDMS and loss of fluidity, resulting in incomplete filling during mold pressing. If the heating temperature is too low or the time is too short, it will cause insufficient adhesion of PDMS and easy tearing during demolding. The present invention preferably pre-cures at 70 - 75°C for 15 - 20 minutes to balance the pre-curing efficiency, adhesion and fluidity.

[0037] In some embodiments of the present invention, in step S3, the pressure applied during mold pressing is 0.5 - 5 N / cm 2 .

[0038] In some preferred embodiments of the present invention, in step S3, the pressure applied during mold pressing is 2 - 4 N / cm 2 .

[0039] In some embodiments of the present invention, in step S3, the mold has a concave microstructure.

[0040] Specifically, during the mold pressing process, if the applied pressure is insufficient, the semi-cured PDMS will not fully fill the mold microstructure, resulting in incomplete microchannels. If the applied pressure is too high, it will cause deformation or fracture of the mold microstructure, or excessive compression of the PDMS layer, resulting in out-of-control thickness. By controlling the pressure applied during mold pressing in the present invention, deformation of the microstructure caused by excessive compression can be avoided, ensuring high-fidelity replication.

[0041] In some embodiments of the present invention, in step S3, the degree of vacuum for vacuum assistance is 10 - 100 kPa.

[0042] In some preferred embodiments of the present invention, in step S3, the degree of vacuum for vacuum assistance is 10 - 20 kPa.

[0043] Specifically, when vacuum-assisted semi-cured PDMS fills the mold microstructure, if the degree of vacuum is insufficient, air bubbles will remain in the microstructure, resulting in channel blockage or optical defects. If the degree of vacuum is too high, it may cause the PDMS prepolymer to boil and damage the microstructure.

[0044] In some embodiments of the present invention, in step S4, the conditions for heat curing are selected from any one of the following:

[0045] i) 80 - 120°C, heating for 30 - 60 minutes;

[0046] ii) At room temperature, let it stand for 45 - 48 h.

[0047] In some preferred embodiments of the present invention, in step S4, the conditions for heat curing are heating at 90 - 110 °C for 30 - 40 min.

[0048] Specifically, during heat curing, for low - temperature or short - time curing, PDMS is not fully cross - linked, the mechanical properties of PDMS are not optimal, which easily leads to an increase in the swelling rate and a decrease in tear resistance; if the curing temperature is too high, it is easy to cause thermal deformation of the hard substrate.

[0049] The second aspect of the present invention provides a structured PDMS thin - layer / hard - substrate composite, which is obtained by using the preparation method described in the first aspect of the present invention.

[0050] In some embodiments of the present invention, in the structured PDMS thin - layer / hard - substrate composite, the thickness of the structured PDMS thin - layer is 10 - 300 μm.

[0051] The third aspect of the present invention provides any one of the following applications of the structured PDMS thin - layer / hard - substrate composite described in the second aspect of the present invention:

[0052] A) Preparing a hard - substrate - PDMS film - hard - substrate sandwich - type microfluidic chip;

[0053] B) Preparing a PDMS microfluidic chip integrated with a high - resolution microscopic observation window;

[0054] C) As a PDMS - based microfluidic chip mold.

[0055] In some embodiments of the present invention, the hard - substrate - PDMS film - hard - substrate sandwich - type microfluidic chip is obtained by bonding the structured PDMS thin - layer / hard - substrate composite with another hard substrate; the other hard substrate and the hard substrate in the structured PDMS thin - layer / hard - substrate composite can be the same or different.

[0056] In some embodiments of the present invention, for the PDMS microfluidic chip integrated with a high - resolution microscopic observation window, in the structured PDMS thin - layer / hard - substrate composite, the thickness of the structured PDMS thin - layer is less than 50 μm.

[0057] Compared with the prior art, the beneficial effects of the present invention are:

[0058] 1) The preparation method of the structured PDMS thin layer / hard substrate composite provided by the present invention ensures the uniformity of the PDMS thin layer through static reflux, laying a foundation for the replication of high-precision structures; endows the semi-cured PDMS thin layer with appropriate adhesiveness through pre-curing, avoiding the out-of-control flow during mold pressing; eliminates bubbles and realizes the complete filling of microstructures through vacuum pressing, ensuring the integrity of the pattern; omits the transfer step through one-piece molding, avoiding the tearing risk caused by the low mechanical strength of the thin film; through the synergistic effect of each step, a structured PDMS thin layer / hard substrate composite with high stiffness, low water volatilization, solvent resistance swelling and microscopic compatibility is obtained, meeting the application requirements of PDMS microfluidic devices in different scenarios;

[0059] 2) The preparation method of the structured PDMS thin layer / hard substrate composite provided by the present invention forms a structured PDMS thin layer directly on the hard substrate, omitting the steps of peeling, transferring and bonding the PDMS thin film in the traditional process, significantly reducing the operation links and improving the production efficiency. It not only reduces the yield loss caused by complex steps, but also reduces the equipment dependence and material waste, reducing the cost, and is applicable to laboratory and industrial production scenarios;

[0060] 3) The preparation method of the structured PDMS thin layer / hard substrate composite provided by the present invention composites the PDMS layer with the hard substrate. The high Young's modulus of the hard substrate (such as glass, silicon wafer) effectively inhibits the deformation of the PDMS layer under fluid pressure, avoiding the collapse or deformation of microchannels, making the composite especially suitable for high-pressure or long-term fluid manipulation scenarios; the one-piece molding process avoids the mechanical stress during the film transfer process, solves the problem of easy tearing and wrinkling of the ultra-thin PDMS layer (10 - 300 μm), and ensures the integrity of the microstructure;

[0061] 4) The preparation method of the structured PDMS thin layer / hard substrate composite provided by the present invention can select glass, silicon wafer, metal sheet or polymer substrate as the hard substrate, and can also select low-cost or high-light-transmitting substrates according to application requirements, with wide material compatibility; the parameters such as pre-curing temperature, vacuum pressure, and mold pressure all provide an optimal range, which can adapt to the high-fidelity replication of different microstructural complexities (such as microchannels, chambers, sensor patterns), and the method has strong scalability.

[0062] 5) The structured PDMS thin layer / hard substrate composite provided by the present invention forms a dense interface by directly bonding the PDMS thin layer to the substrate, reducing the volatilization of moisture through the PDMS material, and is suitable for experiments that require long-term stable humidity, such as cell culture and protein crystallization; the thin geometric characteristics of PDMS itself combined with the hard substrate provide physical support, limiting the degree of swelling and deformation of PDMS in organic solvents, and expanding the application range of microfluidic chips in the biochemical field; by selecting a thin transparent substrate (such as a cover glass) and controlling the thickness of the PDMS thin layer (such as ≤50 μm), the prepared microfluidic chip can adapt to the short working distance (0.2 - 0.3 mm) of a high-magnification objective lens, achieving high-resolution microscopic imaging and meeting the requirements of single-cell observation or nanoscale structure analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 It is a schematic flow chart of the preparation method of the structured PDMS thin layer / hard substrate composite of the present invention;

[0064] Figure 2 It is a schematic flow chart of the preparation of the structured PDMS thin layer / glass slide composite and the digital PCR chip in Example 1;

[0065] Figure 3 It is a schematic flow chart of the preparation of the structured PDMS thin layer / glass slide composite and the single-cell analysis chip in Example 2;

[0066] Figure 4 It is a schematic flow chart of the preparation of the structured PDMS thin layer / high-transmission cover glass composite and the high-resolution microscopic observation chip in Example 3;

[0067] Figure 5 It is a typical microscopic photograph of the high-resolution microscopic observation chip based on the structured PDMS thin layer / high-transmission cover glass composite in Example 3 applied to the observation of the growth of Hela cells under the action of a cisplatin concentration gradient;

[0068] Figure 6 It is a schematic flow chart of the preparation of the structured PDMS thin layer / / polished aluminum plate composite and the PMMA microlens array in Example 4. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0069] The content of the present invention will be further described in detail below through specific embodiments. The raw materials, reagents or devices used in the embodiments can be obtained from conventional commercial channels or can be obtained by existing technical methods without special instructions. Unless otherwise specified, the test or measurement methods are conventional methods in the art.

[0070] Figure 1 It is a schematic flow chart of the preparation method of the structured PDMS thin layer / hard substrate composite of the present invention. The following will be combined withFigure 1 Describe the preparation process of the structured PDMS thin layer / hard substrate composite.

[0071] Example 1

[0072] In this example, a structured PDMS thin layer / glass slide composite is prepared and used to fabricate a digital PCR chip (a glass slide-PDMS film-glass slide sandwich microfluidic chip). Figure 2 Schematic diagram of the preparation process of the structured PDMS thin layer / glass slide composite and the digital PCR chip in Example 1:

[0073] The preparation steps of the structured PDMS thin layer / glass slide composite are as follows:

[0074] S11. Take a glass slide with a thickness of 1 mm (size 75 mm × 25 mm) as the hard substrate, and treat its surface with oxygen plasma (power 50 W, time 1 min) to improve the adhesion of PDMS; mix the silicone polymer and the curing agent at a mass ratio of 10:1, stir for 5 min and perform degassing treatment (vacuum degree 50 kPa, time 30 min) to obtain the PDMS prepolymer (Sylgard184).

[0075] S12. Drop about 500 μL of the PDMS prepolymer on the surface of the glass slide, and manually tilt and rotate the glass slide to make the PDMS prepolymer evenly coated on its surface with a coating thickness of about 200 μm. Let it stand at room temperature (25 °C) for 30 min, and use the surface tension to make the PDMS prepolymer naturally reflux to form a uniform thin layer.

[0076] S21. Place the coated substrate on a 75 °C hot plate and heat for 15 min to form a semi-cured PDMS thin layer.

[0077] S31. Align the silicon-based SU-8 lithography mold (microcavity array design: diameter 100 μm, depth 80 μm, array density 3170 / cm 2 ) with the semi-cured PDMS thin layer, apply a pressure of 5 N / cm 2 Press the mold together, and then place the assembly in a vacuum chamber (vacuum degree 10 kPa) for 30 min to remove air bubbles and ensure that the semi-cured PDMS completely fills the microcavity structure.

[0078] S41. Place the assembly in an oven at 100 °C and cure for 30 min to make the PDMS fully crosslinked. After cooling to room temperature, slowly peel off the mold along the edge to obtain the structured PDMS thin layer / glass slide composite.

[0079] The preparation steps of the digital PCR chip are as follows:

[0080] Align and bond another oxygen plasma-treated (power 50W, time 1min) and perforated glass slide with the structured PDMS thin layer of the structured PDMS thin layer / glass slide composite. Apply a pressure of 1N / cm 2 and heat to 80°C for 30 min to form an irreversible bonding interface, obtaining a digital PCR chip.

[0081] Among them, the total thickness of the digital PCR chip is 2.2 mm, and the thickness of the PDMS film is 200 μm.

[0082] Performance test:

[0083] (1) Stiffness improvement test: Using a universal material testing machine (Instron 5943) and referring to the ASTM D638 standard, conduct a uniaxial tensile test on the structured PDMS thin layer / glass slide composite sample (size 10 mm × 10 mm) with a loading rate of 1 mm / min. Record the stress-strain curve and calculate the elastic modulus. The test results show that the elastic modulus of the structured PDMS thin layer / glass slide composite reaches 60 GPa, which is 4 orders of magnitude higher than that of the full PDMS chip (about 2 MPa), indicating that the composite of the PDMS layer and the high-stiffness hard substrate can effectively inhibit the deformation of the microcavity. Further verification through the microcavity deformation rate test shows that under a fluid pressure of 10 psi, use a laser confocal microscope (Keyence VK-X1000) to scan the cross-sectional morphology of the microcavity and calculate the percentage of the deformation amount to the initial depth. The measured microcavity deformation rate is less than 0.5%, indicating that the composite also has excellent structural stability under high pressure and is suitable for high-pressure environments.

[0084] (2) Solvent resistance test: Immerse the structured PDMS thin layer sample (size 10 mm × 10 mm × 0.2 mm) in paraffin oil (Sigma-Aldrich, density 0.85 g / cm 3 ) and let it stand at a constant temperature of 25°C for 24 h. After taking it out, blot the surface oil stain with filter paper and use a high-precision balance (Mettler Toledo XPE205, accuracy 0.01 mg) to measure the mass change before and after immersion. Calculate the swelling rate according to the formula: Swelling rate = (m wet -m dry ) / m dry ×100%, where m wet is the mass after immersion, and m dry is the mass before immersion. The calculated swelling rate of the structured PDMS thin layer is only 1%, which is much lower than that of the traditional PDMS chip (greater than 20%), indicating that the composite structure of the structured PDMS thin layer and the hard substrate constraint physically limits the structured PDMS thin layer in the swelling direction and can only expand limitedly in the direction perpendicular to the substrate.

[0085] (3) Low moisture volatility test: Inject the fluorescent probe solution (10 μL, containing 0.1 mmol / L FITC) into the microcavity of the structured PDMS thin film / glass slide composite, seal it, and place it in a thermostatic and humidistatic chamber (25 °C, humidity 60%). Use a fluorescence spectrophotometer (Hitachi F-7000) to detect the fluorescence intensity of the solution every 24 h (excitation wavelength 490 nm, emission wavelength 520 nm). Calculate the volume loss of the solution through the standard curve. The results show that the moisture volatilization loss of the structured PDMS thin film / glass slide composite is less than 2% after 72 h, indicating that the glass substrate is beneficial for blocking the penetration of water molecules, reducing the moisture volatility of the PDMS material, and being more suitable for long-term experiments.

[0086] (4) Digital PCR detection of viral RNA: Synthesize a standard product of a certain viral RNA (concentration gradient: 0.1 - 100 copies / μL), extract RNA using the QIAamp Viral RNA Mini Kit, load the sample onto the digital PCR chip, and use an Eppendorf flat PCR instrument ( nexus flat) for amplification (cycle parameters: 95 °C for 10 min → 45 cycles of 95 °C for 20 s → 60 °C for 40 s). Calculate the copy number by counting the number of positive microcavities. The sensitivity test shows that the lowest detection limit is 2 copies / mL, and the coefficient of variation (CV) of the repeatability test (n = 10) is less than 5%, indicating that the non-deformation of the microcavity ensures the uniform distribution of nucleic acids and improves the detection accuracy.

[0087] The microcavity-type digital PCR chip relies on a high-density microcavity array to discretely amplify nucleic acid samples, requiring the microfluidic chip to have the characteristics of high stiffness, low deformation, low moisture volatility, and anti-solvent swelling. Traditional PDMS chips are difficult to meet the requirements of accurate and reliable nucleic acid detection in digital PCR technology due to their large thickness, insufficient stiffness, and serious moisture volatilization. Based on the preparation method of the structured PDMS thin film / hard substrate composite provided by the present invention, a structured PDMS thin film is directly formed on the hard substrate, and combined with the sandwich bonding process, a highly reliable microcavity array-type digital PCR chip is successfully prepared, meeting the application requirements of PDMS materials in high-precision digital PCR scenarios.

[0088] Example 2

[0089] In this example, a structured PDMS thin film / glass slide composite is prepared and used to prepare a single-cell analysis chip (a slide-PDMS film-slide sandwich-type microfluidic chip). Figure 3 Schematic diagram of the preparation process of the structured PDMS thin film / glass slide composite and the single-cell analysis chip in Example 2:

[0090] The preparation steps of the structured PDMS thin film / glass slide composite are as follows:

[0091] S11. Take a glass slide with a thickness of 1 mm (size 75 mm × 25 mm) as the hard substrate, and the surface is treated by oxygen plasma (power 50 W, time 1 min) to improve the adhesion of PDMS; mix the silicone polymer and the curing agent at a mass ratio of 10:1, stir for 5 min and perform degassing treatment to obtain the PDMS prepolymer (Sylgard 184);

[0092] S12. Use an automatic coater to coat the PDMS prepolymer on the surface of the glass slide at a rotation speed of 200 rpm, with a coating thickness of about 180 μm, and let it stand at room temperature (25 °C) for 20 min. Eliminate the edge accumulation effect through surface tension to form a uniform PDMS thin film with a thickness of about 150 μm;

[0093] S21. Place the coated substrate on an 85 °C hot plate and heat for 10 min to form a semi-cured PDMS thin film;

[0094] S31. Spray fluorosilane (1H,1H,2H,2H-perfluorooctyltrichlorosilane) on the surface of the electroformed nickel mold (sample cavity: depth 100 μm, width: 2 cm, length: 5 cm; depth microcavity design: diameter 15 μm, depth 10 μm, array density 9055 / cm 2 and the cavity spacing is 5 μm) as a release agent, apply a pressure of 5 N / cm 2 Press the mold onto the surface of the semi-cured PDMS thin film, and then place the assembly in a vacuum chamber (vacuum degree 5 kPa) and process for 20 min to ensure that the semi-cured PDMS completely fills the mold microstructure;

[0095] S41. Place the assembly in an oven at 90 °C and cure for 50 min to completely crosslink the PDMS. After cooling to room temperature, slowly peel off the mold along the edge to obtain the structured PDMS thin film / glass slide composite.

[0096] The preparation steps of the single-cell analysis chip are as follows:

[0097] After another glass slide is treated by ultraviolet ozone (wavelength 185 nm, intensity 30 mW / cm 2 and time 5 min), align and bond it with the structured PDMS thin film of the structured PDMS thin film / glass slide composite, apply a pressure of 0.5 N / cm 2 Press and heat to 90 °C and maintain for 15 min to form an irreversible bonding interface to obtain the single-cell analysis chip.

[0098] Among them, the total thickness of the single-cell analysis chip is 2.1 mm, the thickness of the PDMS film is 100 μm, and the overall thickness of the sample cavity is uniform.

[0099] Performance test:

[0100] (1) Stiffness improvement test: The test method is the same as that in Example 1. The result shows that the elastic modulus of the structured PDMS thin layer / glass slide composite in Example 2 is 62 GPa.

[0101] (2) Deformation inhibition test: Use a compressed carbon dioxide gas cylinder and a digital pressure gauge to conduct a mechanical property test on the structured PDMS thin layer / glass slide composite. Seal the chip outlet, open the pressure reducing valve of the gas cylinder, apply a pressure of 10 psi to the inside of the chip, and maintain for 10 min. After the pressure is applied, use a laser confocal microscope to perform a three-dimensional topography scan on the cross-section of the sample cavity to obtain the original data (unpressurized state) and the data after pressurization of the sample cavity depth, and calculate the deformation rate. The calculation formula is: Deformation rate = ∣D loaded -D initia l∣D initial ×100%, where D initia l is the initial sample cavity depth (100 μm), and D loaded is the depth after pressurization. The result shows that the average deformation rate of the microcavity of the composite under a fluid pressure of 10 psi is only 0.07% (n = 10 repeated tests), and the standard deviation is ±0.5%, indicating that the composite has high stiffness characteristics and can effectively avoid the problem of sample cavity collapse of traditional PDMS chips.

[0102] (3) Capture efficiency test: Use a HeLa cell suspension (density 1×10 5 cells / mL, medium is DMEM containing 10% FBS), calibrate the cell concentration through a flow cytometer (BD Accuri C6) to ensure the uniformity of the suspension. Inject the cell suspension into the inlet of the single-cell analysis chip at a flow rate of 5 μL / min, and let it stand at 25 °C for 30 min to allow the cells to settle into the microcavities. Use an inverted fluorescence microscope (Nikon Eclipse Ti2, objective 20× NA 0.45), combined with an automatic stage (Prior H117E), randomly select 10 fields of view (each field of view contains 100 - 120 microcavities), and a total of 1000 microcavities are counted. Record bright field and fluorescence images (Calcein-AM stained live cells, Ex / Em = 488 / 517 nm) through NIS-Elements AR software. Count the proportion of microcavities containing a single fluorescence signal (area > 50 μm 2 and intensity threshold > 2000 AU), and count the proportion of microcavities without fluorescence signals as the cavity rate. The result shows that the single-cell capture efficiency of the single-cell analysis chip reaches 96.5% (±1.2%, n = 3 independent experiments), and the cavity rate < 10%, which is significantly better than traditional PDMS chips (capture efficiency is about 85%, cavity rate > 20%).

[0103] Single-cell analysis requires the capture and isolation of single cells through a high-density microcavity array. Conventional single-cell analysis chips with a microcavity array often integrate a high-density microcavity array under a large sample cavity. Since the sample cavity has a flat structure (aspect ratio < 0.01), for traditional PDMS chips, due to insufficient stiffness, the low-aspect-ratio sample cavity is extremely prone to collapse, resulting in a decrease in cell capture efficiency. Based on the preparation method of the structured PDMS thin layer / hard substrate composite provided by the present invention, a structured PDMS thin layer is directly formed on the hard substrate, and combined with the sandwich bonding process, the stable formation of a high-density microcavity array type single-cell analysis chip and efficient single-cell encapsulation are realized.

[0104] Example 3

[0105] In this example, a structured PDMS thin layer / high-transparency cover glass composite is prepared and used to prepare a high-resolution microscopic observation chip (a PDMS microfluidic chip integrated with a high-resolution microscopic observation window). Figure 4 The following is a schematic diagram of the preparation process of the structured PDMS thin layer / high-transparency cover glass composite and the high-resolution microscopic observation chip in Example 3:

[0106] The preparation steps of the structured PDMS thin layer / high-transparency cover glass composite are as follows:

[0107] S11. Take a high-transparency cover glass with a thickness of 0.17 mm (size 24 mm × 60 mm) as the hard substrate, and the surface is treated with oxygen plasma (power 30 W, time 2 min) to improve the adhesion of PDMS; mix the silicone polymer and the curing agent at a mass ratio of 10:1, stir for 5 min and perform degassing treatment (vacuum degree 20 kPa, time 20 min) to obtain a PDMS prepolymer (Sylgard184).

[0108] S12. Use the spin-coating process (500 rpm / 10 s → 1500 rpm / 30 s) to form a PDMS prepolymer layer with an initial thickness of about 80 μm on the surface of the cover glass, and let it stand at room temperature (25 °C) for 30 min to eliminate the edge accumulation effect through surface tension and form a uniform PDMS thin layer with a thickness of about 60 μm.

[0109] S21. Place the coated substrate at room temperature overnight (about 12 h) to form a semi-cured PDMS thin layer.

[0110] S31. Align the silicon-based SU-8 lithography mold (microcavity array design: diameter 100 μm, depth 80 μm, array density 3170 / cm 2 ) with the semi-cured PDMS thin layer and apply a pressure of 1.2 N / cm 2Press the mold together, and then place the assembly in a vacuum chamber (vacuum degree 15 kPa) for 20 min to ensure that the semi-cured PDMS completely fills the gradient channels;

[0111] S41. Place the assembly in an oven at 100 °C for 30 min to completely crosslink the PDMS. After cooling to room temperature, slowly peel off the mold along the edge to obtain a structured PDMS thin layer / high-transmission cover glass composite.

[0112] The preparation steps of the high-resolution microscopic observation chip are as follows:

[0113] Graft polylysine (0.1 mg / mL, pH = 7.4) on the surface of the PDMS gradient channels to enhance cell adhesion. Align a perforated glass slide with the structured PDMS thin layer of the structured PDMS thin layer / high-transmission cover glass composite after oxygen plasma activation, and apply a pressure of 1.2 N / cm 2 , heat to 90 °C and maintain for 30 min to form an irreversible bonding interface, thus obtaining the high-resolution microscopic observation chip.

[0114] Among them, the high-resolution microscopic observation chip is a thin-bottom closed concentration gradient chip, and the thickness of the bottom layer of the chip is 0.23 mm.

[0115] Performance test:

[0116] (1) Microscopic compatibility test: Use an inverted confocal microscope (Leica TCS SP8, equipped with a 63× oil immersion objective lens, NA 1.4, working distance 0.3 mm), and use a USAF 1951 resolution test target (Edmund Optics, model 58-198). Fix it on the surface of the structured PDMS thin layer / high-transmission cover glass composite, and immerse the objective lens and the sample through glycerol (refractive index 1.47) for matching. Under the excitation of a 532 nm laser, scan the line pair structure of the 6th unit in the 7th group (theoretical resolution 0.2 μm), adjust the Z-axis focus to the maximum contrast, and record the minimum line width that can be clearly distinguished. The thickness of the structured PDMS thin layer in the structured PDMS thin layer / high-transmission cover glass composite is 60 μm, which is adapted to the working distance of the objective lens (0.3 mm). The line pair structure of the 6th unit in the 7th group of the USAF test target is clearly imaged (contrast > 90%), verifying that the imaging resolution reaches 0.2 μm, while the traditional PDMS chip (thickness 1.5 mm) cannot be focused because it exceeds the working distance of the objective lens.

[0117] (2) Gradient stability test: Using a dual-channel syringe pump (Harvard Apparatus PHD Ultra), inject FITC-dextran (10 kDa, 1 mg / mL) solution and PBS buffer into the structured PDMS thin layer / high-transparency cover glass composite at a flow rate of 0.5 μL / min respectively to generate a linear concentration gradient. The fluorescence intensity in the channel (excitation wavelength 488 nm, emission wavelength 517 nm) was collected every 30 min by a confocal microscope (Leica TCS SP8). Ten equally spaced points were selected along the channel length direction, and the intensity values were recorded. The gradient stability was evaluated by the normalized standard deviation of intensity (NSD), and the calculation formula is: NSD = σ / μ × 100%, where σ is the standard deviation of the intensity values at 10 positions at each time point, and μ is the average value. The results showed that NSD < 5% within 6 h, while NSD > 15% after 3 h for the traditional PDMS chip, indicating that the gradient of the structured PDMS thin layer / high-transparency cover glass composite prepared by the present invention has no obvious diffusion or deformation and has the advantage of gradient stability.

[0118] (3) Cell dynamic observation test: HeLa cells were seeded on the surface of the PDMS gradient channel of the high-resolution microscopic observation chip (density 5×10 4 cells / cm 2 ), and the culture medium was DMEM containing 10% FBS. The cells were cultured at 37 °C and 5% CO2 for 24 h. A cisplatin concentration gradient (0 - 100 μmol / L) was generated at a flow rate of 0.2 μL / min by a syringe pump and maintained for 6 h. Using a live cell imaging system (Nikon BioStudio-T, environmental control: 37 °C, 5% CO2), bright field and fluorescence images were collected every 10 min (mitochondria were labeled with the mitochondrial protein TOM20 antibody). The degree of mitochondrial fragmentation was analyzed by NIS-Elements AR software (morphological score: 0 = intact network, 4 = completely fragmented). Cells in the high-concentration cisplatin region (50 - 100 μmol / L) had a mitochondrial score > 3 within 4 h, showing significant apoptosis characteristics; cells in the low-concentration region (0 - 10 μmol / L) had a score < 1, indicating a good cell survival state. Figure 5 Figure for the typical microscopic photograph of the high-resolution microscopic observation chip based on the structured PDMS thin layer / high-transparency cover glass composite in Example 3 applied to the observation of the growth of Hela cells under the action of a cisplatin concentration gradient. It can be seen from Figure 5 that the high-resolution microscopic observation chip based on the structured PDMS thin layer / high-transparency cover glass composite can achieve a dynamic resolution up to the sub-organelle level, and the morphological changes of mitochondria are clearly distinguishable.

[0119] High-resolution microscopic observation of the effect of drug gradients on cell behavior requires the chip to have optical transparency, an ultra-thin substrate (≤200 μm), and stable gradient generation ability. Traditional PDMS chips cannot adapt to the short working distance (0.2 - 0.3 mm) of high-magnification objective lenses due to their excessive thickness (>1 mm), and solvent diffusion easily causes gradient instability. Based on the preparation method of the structured PDMS thin layer / hard substrate composite provided by the present invention, in this example, a cover glass is used as the hard substrate, combined with the design of micron-scale gradient channels, to prepare a thin-bottom chip that can be directly placed on the objective lens of an inverted microscope, realizing high-resolution real-time observation of cell dynamics under the action of drug gradients.

[0120] Example 4

[0121] In this example, a structured PDMS thin layer / polished aluminum plate composite is prepared and directly used as a hot embossing mold for the preparation of PMMA microlens arrays. Figure 6 Schematic diagram of the preparation process of the structured PDMS thin layer / / polished aluminum plate composite and PMMA microlens arrays in Example 4:

[0122] S11. Take a polished aluminum plate with a thickness of 2 mm (size 100 mm × 100 mm) as the hard substrate, and form a porous alumina layer on the surface by anodic oxidation treatment (voltage 20 V, time 30 min) to enhance the PDMS anchoring force; mix the silicone polymer and the curing agent in a mass ratio of 5:1, stir for 5 min and perform degassing treatment (vacuum degree 20 kPa, time 30 min) to obtain the PDMS prepolymer.

[0123] S12. Use the doctor blade coating process to form a PDMS prepolymer layer with an initial thickness of 300 μm on the surface of the polished aluminum plate, and let it stand at room temperature (25 °C) for 25 min. Eliminate the coating fluctuations by gravity leveling to form a uniform PDMS thin layer with a thickness of about 250 μm.

[0124] S21. Place the coated substrate at room temperature overnight (about 12 h) to form a semi-cured PDMS thin layer. Reduce the shrinkage rate of PDMS during the curing process by low-temperature curing to ensure the accuracy of the spacing between micro-lenses in the microlens array.

[0125] S31. Use AZ4620 positive photoresist to fabricate a micro-cylindrical array structure on a silicon wafer. Through the thermal reflux method, fabricate a spherical cap-shaped microlens array structure (microlens design: curvature radius 25 μm, array period 50 μm, height 18 μm). Align the spherical cap-shaped microlens array structure with the semi-cured PDMS thin layer and apply a pressure of 5 N / cm 2 Press the mold together, and then place the combination in a vacuum chamber (vacuum degree 50 kPa) for 20 min to ensure that the semi-cured PDMS completely fills the micro-structure of the mold.

[0126] S41. Place the composite at room temperature and let it stand for 48 h to completely cure the PDMS. Then peel off the master mold to obtain a structured PDMS thin layer / polished aluminum plate composite.

[0127] The preparation steps of the PMMA microlens array are as follows:

[0128] Using the structured PDMS thin layer / polished aluminum plate composite as the aluminum-PDMS composite mold, heat a 1-mm-thick PMMA sheet to 135 °C (30 °C higher than its glass transition temperature), then place the aluminum-PDMS mold on its surface, apply a pressure of 8 N / cm 2 and keep it for 5 min to completely fill the PMMA microlens concave mold. After cooling to 60 °C, demold to obtain the PMMA microlens array.

[0129] Performance test:

[0130] (1) Mold accuracy test: Use an atomic force microscope (AFM, Bruker Dimension Icon, Tap150 probe) to perform three-dimensional scanning on the microlens morphologies of the aluminum-PDMS composite mold and the original silicon-based master mold. The scanning range is 50 μm × 50 μm, the resolution is 512 × 512 pixels, the scanning rate is 1 Hz, and the probe elastic coefficient is 5 N / m. Extract the radius of curvature (R 母模 = 50 ± 0.3 μm) and the array period (P 母模 = 25 ± 0.2 μm) of the master mold and the PDMS mold through NanoScope Analysis software, and calculate the relative errors: curvature error = ∣R 模具 - R 母模 ∣ / R 母模 × 100%, period error = ∣P 模具 - P 母模 ∣ / P 母模 × 100%. The results show that the curvature error of the aluminum-PDMS composite mold is less than 0.8% (R 模具 = 50.2 ± 0.2 μm), and the period error is less than 0.6% (P mold = 24.8 ± 0.15 μm), meeting the λ / 10 optical surface accuracy requirements.

[0131] (2) Demolding simplicity test: Use a universal material testing machine (Instron 5943) to refer to the ASTM D3330 tape peeling standard. Customize a fixture to fix the interface between the PMMA sheet and the aluminum-PDMS composite mold, and separate it vertically at a rate of 5 mm / min, and record the maximum peeling force. After demolding, scan the surface of the PMMA microlens with a white light interferometer (Zygo NewView 9000) to detect scratches or residual defects. The results show that the average demolding force is 0.15 N / cm 2, much lower than that of traditional metal molds (greater than 2 N / cm 2 ), the surface roughness Ra of PMMA is less than 5 nm, and there is no visible damage. Due to the low surface energy characteristics of the PDMS material, it is not easy to interact or adhere with other materials. Therefore, when the structured PDMS thin layer / polished aluminum plate composite provided by the present invention is directly used as a PDMS-based mold, it is easy to demold after hot embossing without complex mold surface treatment.

[0132] (3) Optical performance test: Use a helium-neon laser (wavelength 632.8 nm, power 1 mW) to vertically irradiate the microlens array, and measure the focal spot intensity distribution through a beam quality analyzer (Thorlabs BP209) to calculate the focusing efficiency: Focusing efficiency = I focal / I total ×100%, where I focal is the light intensity integral of the focal region (diameter 3 × Airy disk), and I total is the total incident light intensity. Use a confocal displacement sensor (Keyence LT-9010M) to select 20 microlenses along the array to measure the deviation between the actual focal length and the designed value (f design = 75 μm). The results show that the focusing efficiency > 92% (wavelength 632.8 nm), and the CV value of the focal length consistency < 1.3% (favg = 74.8 ± 0.9 μm, n = 20).

[0133] (4) Cost-benefit analysis: Statistically analyze the cost of single mold production (materials, equipment depreciation, labor), and compare it with the traditional lithography process. It is found that the cost of single mold production is reduced to 20% of that of silicon-based molds, which is suitable for mass production.

[0134] Optical microlens arrays (MLAs) are widely used in the fields of imaging, optical communication, and integrated photonics. Their preparation relies on high-precision molds to achieve nanoscale surface topography control. The production of traditional metal or silicon-based molds requires complex lithography and etching processes, with high costs and difficult demolding. Based on the preparation method of the structured PDMS thin layer / hard substrate composite provided by the present invention, this embodiment utilizes the high-precision microstructure replication ability of the structured PDMS thin layer / polished aluminum plate composite to directly serve as a hot embossing mold, combined with the molding characteristics of thermoplastic polymers (PMMA), to achieve low-cost and high-consistency batch production of optical microlens arrays.

Claims

1. A preparation method of a structured PDMS thin layer / rigid substrate composite, characterized in that, It includes the following steps: S1. Coating the PDMS prepolymer on the surface of the hard substrate, standing still and refluxing to form a uniform PDMS thin layer; S2. Performing pre-curing treatment on the PDMS thin layer to obtain a semi-cured PDMS thin layer; S3. Pressing the mold onto the surface of the semi-cured PDMS thin layer, and using vacuum assistance to fill the microstructures of the mold with the semi-cured PDMS; S4. Heating and curing, and peeling off the mold to obtain the structured PDMS thin layer / hard substrate composite.

2. The preparation method according to claim 1, wherein The hard substrate is selected from glass, silicon wafer, metal sheet or polymer substrate.

3. The preparation method according to claim 1, characterized in that In step S1, in the PDMS prepolymer, the mass ratio of the silicon-based polymer to the curing agent is (5-20):

1.

4. The preparation method according to claim 1, characterized in that, In step S1, the time of standing still and refluxing is 10-20 min.

5. The preparation method according to claim 1, characterized in that, In step S2, the conditions of the pre-curing treatment are selected from any one of the following: a) 70-75 °C, heating for 15-20 min; b) 80-85 °C, heating for 10-15 min; c) 90-100 °C, heating for 5-10 min; d) At room temperature, standing still for 10-12 h.

6. The preparation method according to claim 1, characterized in that, In step S3, the pressure applied by the die pressing is 0.5 - 5 N / cm 2 ; the degree of vacuum for the vacuum assistance is 10 - 100 kPa.

7. The preparation method according to claim 1, characterized in that, In step S4, the conditions of the heating and curing are selected from any one of the following: i) 80-120 °C, heating for 30-60 min; ii) At room temperature, standing still for 45-48 h.

8. A structured PDMS thin layer / hard substrate composite, characterized in that, It includes being obtained by using the preparation method described in any one of claims 1-7.

9. The structured PDMS thin layer / hard substrate composite according to claim 8, wherein, In the structured PDMS thin layer / hard substrate composite, the thickness of the structured PDMS thin layer is 10-300 μm.

10. Any application of the structured PDMS thin layer / hard substrate composite described in claim 8 or 9 in the following aspects: A) Preparing a hard substrate-PDMS film-hard substrate sandwich microfluidic chip; B) Preparing a PDMS microfluidic chip integrated with a high-resolution microscopic observation window; C) As a PDMS-based microfluidic chip mold.

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