Glass embedded detachable surface wave acoustic fluidic sorting chip and manufacturing method thereof

Abstract

The present invention discloses a glass-embedded detachable surface wave acoustic fluidic sorting chip and a manufacturing method thereof. The sorting chip comprises an upper glass-embedded PDMS substrate and a lower piezoelectric transducer cover. A PDMS coupling layer is contained between the substrate and the cover. The glass-embedded PDMS substrate is formed by a secondary inverted mold method. The glass-embedded detachable surface wave acoustic fluidic sorting chip provided by the present invention can meet the disposable use requirements of biological sample processing devices. At the same time, the sound field intensity is enhanced by the glass sheet to ensure a better sorting effect for submicron particles such as exosomes. The manufacturing method of the glass-embedded detachable surface wave acoustic fluidic sorting chip provided by the present invention can ensure the uniformity of the height of the sorting channel while embedding the glass sheet into the top of the sorting channel to enhance the sound field, and will not form a stepped fault at the transition interface between the glass material and the PDMS material, thereby ensuring the stability of the flow field in the channel.

Description

Technical Field

[0001] The invention relates to the field of microfluidics and biological particle sorting technology, and in particular to a glass-embedded detachable surface wave acoustic fluidics sorting chip and a manufacturing method thereof. Background Art

[0002] Purification and separation of biological particles are basic pretreatment steps in biomedical and biochemical analysis, where enriched samples can improve diagnostic and therapeutic effects. Since extracellular vesicles (EVs) carry important molecular information, their efficient sorting has received increasing attention. Currently, a variety of biological particle separation technologies and devices have been widely studied. Among them, the acoustic fluidic sorting chip that combines surface acoustic wave (SAW) with microfluidics technology for particle separation in trace samples is a sorting carrier with good biocompatibility, high efficiency, simple structure, and high controllability, and has broad application prospects. The surface acoustic wave acoustic fluidic device mainly uses lithium niobate (LiNbO 3 ) piezoelectric single crystal is used as the cover, and polydimethylsiloxane (PDMS) microfluidic chip is used as the substrate. Among them, the lithium niobate piezoelectric single crystal contains a finely structured interdigital transducer, and its processing process is complicated and costly. In addition, the substrate and cover are irreversibly bonded by plasma treatment, which faces high costs when used for one-time processing of biological samples. Studies have shown that adding a PDMS coupling layer between lithium niobate and the PDMS chip can achieve detachable bonding between the PDMS chip and lithium niobate, while ensuring the effective radiation of surface acoustic waves and meeting the bonding pressure requirements brought by the sample during the sorting process. Among them, the PDMS chip substrate and the film form an irreversible bond after plasma treatment, and the lithium niobate cover and the film form a reversible bond through van der Waals force to achieve convenient replacement of the PDMS chip during biological sample processing and reuse of the lithium niobate substrate with interdigital electrodes. However, the presence of the PDMS coupling layer is bound to lead to the loss of acoustic energy. Compared with conventional surface wave acoustic fluidic devices, its acoustic field distribution and acoustic pressure will change greatly, which will have a greater negative impact when used for particle sorting. Especially when applied to the sorting of submicron particles such as exosomes, sufficient acoustic pressure amplitude and acoustic radiation force are the key to achieving effective sorting. When using a detachable surface wave acoustic fluidic device, a higher input power is required, which will greatly increase the risk of lithium niobate substrate fragmentation. Based on this, it is necessary to design a detachable, disposable surface wave acoustic fluidic sorting chip that can ensure effective sorting. Summary of the invention

[0003] In view of the above-mentioned shortcomings, considering that the disposable acoustic fluidic chip formed by bonding the PDMS coupling layer to the PDMS chip has the loss of sound propagation in the bottom PDMS coupling layer and the loss of PDMS absorption at the top of the sorting channel, the present invention provides a glass-embedded detachable surface wave acoustic fluidic sorting chip and a method for making the same. The glass-embedded detachable surface wave acoustic fluidic sorting chip prepared by this method embeds a glass sheet at the top of the sorting channel to reduce the absorption of acoustic energy by PDMS, enhance the reflection of acoustic energy, and meet the required sound pressure intensity in the channel at a lower power, greatly reducing the risk of lithium niobate substrate fragmentation. The glass-embedded detachable surface wave acoustic fluidic sorting chip provided by the present invention meets the requirement of disposable use, while enhancing the local sound field intensity through the glass sheet, compensating to a certain extent for the sound energy lost in the disposable acoustic fluidic chip due to the PDMS coupling layer, so that the sorting has a better effect.

[0004] In order to achieve the above-mentioned purpose, the present invention provides a glass-embedded detachable surface wave acoustic fluidic sorting chip and a method for manufacturing the same. A glass-embedded detachable surface wave acoustic fluidic sorting chip comprises a glass-embedded PDMS substrate, a PDMS coupling layer and a piezoelectric transducer cover. The glass-embedded PDMS substrate comprises a microchannel structure, in which a glass sheet is partially embedded in the sorting channel for local enhancement of the sound field; the glass-embedded PDMS substrate and the PDMS coupling layer are irreversibly bonded by oxygen plasma treatment. The PDMS coupling layer and the piezoelectric transducer cover are reversibly bonded by van der Waals force to achieve a detachable connection between the substrate and the cover.

[0005] It should be noted that, in order to address the problem that the PDMS material of the PDMS coupling layer of the current disposable acoustofluidic sorting chip weakens the acoustic field in the sorting channel, the present invention adds a glass sheet with a higher impedance coefficient on the top of the sorting channel to reduce the absorption of acoustic energy by the PDMS on the top of the sorting channel and reflect more acoustic energy, thereby enhancing the sound pressure inside the sorting channel, reducing the required input power, and reducing the risk of rupture of the interdigital transducer substrate.

[0006] The microchannel structure is located on a glass-embedded PDMS substrate, and includes a sheath flow inlet 1, a sample inlet, a sheath flow inlet 2, a sheath flow channel 1, a sample channel, a sheath flow channel 2, a sorting channel, a glass sheet, a large cell channel, an exosome channel, a large cell outlet, and an exosome outlet; the sheath flow channel 1, the sample channel, and the sheath flow channel 2 are connected at the inlet and outlet of the sorting channel; the large cell channel and the exosome channel are connected at the outlet of the sorting channel; and the glass sheet is embedded in the top of the sorting channel.

[0007] The glass-embedded PDMS substrate is also provided with a sheath flow inlet 1, a sample inlet, a sheath flow inlet 2, a large cell outlet, and an external vesicle outlet; the sheath flow inlet 1, the sample inlet, and the sheath flow inlet 2 are respectively connected to an external sampling device; the large cell outlet and the external vesicle outlet are respectively connected to an external collecting device.

[0008] The main part of the glass-embedded PDMS substrate and the PDMS coupling layer are both made of PDMS material, and are pressed together to form an irreversible bond after plasma treatment, thereby becoming a disposable whole; the PDMS coupling layer is a PDMS film with a thickness of 30-50 μm, and after forming an irreversible bond with the upper cover sheet, the excess part is cut off.

[0009] According to one aspect of the present invention, the depth of the microchannel structure portion is 50-100μm; the diameters of the sheath flow inlet 1, sample inlet, sheath flow inlet 2, large cell outlet, and extracellular vesicle outlet are 1mm; the widths of the sheath flow channel 1 and sheath flow channel 2 are 100-200μm; the width of the sample channel is 300-600μm; the width of the sorting channel is 600-1200μm; the width of the large cell outlet and extracellular vesicle outlet is 500-1000μm; the sheath flow channel 1 and sheath flow channel 2 are symmetrically distributed about the sample channel, and the angle between them and the sample channel is 30-60°; the sample channel is parallel to the sorting channel; the angle between the large cell channel and the extracellular vesicle channel is 60-120°; the angle between the sorting channel and the propagation direction of the surface acoustic wave is 65-75°; the height of the glass sheet is 0.1mm, the width is consistent with the width of the sorting channel, and the length is smaller than the sorting channel.

[0010] It should be noted that a glass-embedded detachable surface wave acoustic fluidic sorting chip is manufactured by a PDMS secondary molding method. The method is different from conventional PDMS molding. Instead, a glass sheet with a width consistent with the sorting channel is attached to the sorting channel part of the PDMS negative mold of the first molding as a template for a second molding.

[0011] It should be noted that, during the molding process, a surface treatment solution needs to be prepared to treat the surface of the negative mold to ensure that the PDMS of the second molding can be separated from the PDMS of the first molding.

[0012] It should be noted that, since PDMS can form reliable reversible bonds with glass, after a customized glass part is affixed to the surface-treated negative mold and then reverse molded, the glass part can be retained in the PDMS of the secondary mold, and since a gapless reversible bond is formed between the glass part and the negative mold, the size of the microstructure in the negative mold is still retained in the microstructure of the PDMS of the secondary mold, and no step-like fault is formed in the plane where the glass and PDMS are located, thereby ensuring the stability of the flow field in the sorting channel and enhancing the sound pressure in the sorting channel.

[0013] The process of secondary molding is generally as follows: First, prepare a negative mold through the general PDMS photolithography molding method. Unlike the general microfluidic chip molding, the structure molded out this time should be used as a template for the secondary molding, and the microchannel part should be protruding. Then, the negative mold is cut and demolded according to the chip size. After pasting the processed glass piece of the corresponding size in the channel, the surface is treated with a prepared surface treatment solution. Next, after cleaning the surface of the negative mold, the PDMS molded once is placed in a container and molded with the prepared PDMS solution. Finally, the glass-embedded PDMS substrate obtained by the secondary molding is cut, demolded, and punched according to the chip size, and the glass-embedded PDMS substrate is bonded to the PDMS coupling layer after plasma treatment.

[0014] It should be noted that the mold uses a PC culture dish as a container, because after PMDS is cured in the PC culture dish, it will not adhere to it and can be easily removed from the PC culture dish and cut. The PDMS curing temperature is set to 75°C and the PDMS curing time is set to 4 hours.

[0015] It should be noted that the surface treatment solution used in the mold casting uses raw materials: 0.2 mol / L disodium hydrogen phosphate solution, 0.1 mol / L citric acid solution and 0.3% HPMC solution in a volume ratio of 1:4:5.

[0016] The beneficial effects of this application are:

[0017] 1. The glass-embedded PDMS substrate and the PDMS coupling layer are plasma treated to form an irreversible bond, and the PDMS coupling layer and the piezoelectric transducer cover are reversibly bonded by van der Waals force, so as to achieve a detachable connection between the substrate and the cover, which is easy to assemble and disassemble, thereby ensuring that the microchannel does not directly contact the piezoelectric substrate, which can ensure that the microchannel can withstand a sufficiently large liquid pressure and can also ensure the disposable use requirements of the glass-embedded PDMS chip with enhanced acoustic field.

[0018] 2. The present application embeds a glass sheet in the microchannel to reduce the attenuation of sound waves at the top of the microchannel and enhance the reflection of sound waves (existing studies have shown that PDMS reflects only about 4% of sound energy, while the glass sheet reflects up to 89%) to compensate for the attenuation of sound energy when the sound wave passes through the PDMS coupling layer, thereby retaining more sound energy for particle manipulation and separating particles in a shorter stroke.

[0019] 3. The present application is designed to embed a glass sheet into a microchannel to enhance the utilization rate of acoustic energy. This cannot be prepared using the usual PDMS microfluidic chip preparation method, and the glass sheet cannot be combined with the photoresist in a seamless and low-connection-force manner. However, the PDMS double casting method can ensure that a layer of tightly contacted glass sheet is directly added to the microchannel, and is demolded together with the PDMS main body during demolding, ensuring that the height of the microchannel does not change after the glass sheet is embedded, and is only determined by the glue leveling height.

[0020] Figures and their brief description

[0021] Figure 1 It is a structural schematic diagram of the present invention;

[0022] Figure 2 A schematic diagram of the structure of a surface acoustic wave device used in the present invention;

[0023] Figure 3 It is a schematic diagram of the PDMS coupling layer structure of the present invention;

[0024] Figure 4 A bottom view of the glass-embedded PDMS substrate of the present invention;

[0025] Figure 5 The PDMS portion in the glass-embedded PDMS substrate of the present invention;

[0026] Figure 6 The glass sheet portion of the glass-embedded PDMS substrate of the present invention;

[0027] Figure 7 It is a schematic diagram of the manufacturing method of the present invention;

[0028] Among them: 1-glass embedded PDMS substrate; 2-PDMS coupling layer; 3-glass sheet; 4-lithium niobate base part of surface acoustic wave device; 5-interdigitated electrode part of surface acoustic wave device; 6-sheath flow inlet 1; 7-sample inlet; 8-sheath flow inlet 2; 9-large cell outlet; 10-extravesicle outlet; 11-sheath flow channel 1; 12-sample channel; 13-sheath flow channel 2; 14-sorting channel; 15-large cell channel; 16-extravesicle channel. DETAILED DESCRIPTION

[0029] The present invention is further described below in conjunction with the accompanying drawings. The accompanying drawings are only used for exemplary descriptions and are not physical drawings, and should not be construed as limiting the present invention. In order to better describe the specific implementation methods of the present invention, some components in the accompanying drawings are appropriately scaled and are not proportional to the physical objects; for those skilled in the art, some well-known steps in the accompanying drawings are not described in detail, and the focus is on the innovative parts of the present invention.

[0030] Example 1: Figure 3 As shown, a method for manufacturing a glass-embedded PDMS chip with acoustic field enhancement comprises the following steps:

[0031] Step 1: Use silicon wafer as substrate and SU 8 as photoresist to prepare a negative mold.

[0032] Step 2: Place the negative mold in a flat-bottomed PC culture dish. Prepare the PDMS solution with a mass ratio of PDMS prepolymer: curing agent = 10:1, and pour the PDMS solution into the culture dish so that the PDMS liquid level is about 5 mm.

[0033] Step 3: Place the PC culture dish containing the PDMS solution in a vacuum vessel, and ensure that the bottom is vacuumed horizontally. The vacuuming time should be long enough to ensure that the bubbles in the PDMS solution disappear.

[0034] Step 4: Place the vacuumed vessel containing the PDMS solution on a hot plate for curing. The curing temperature is set at 75°C and the curing time is 4 hours.

[0035] Step 5: Use a hand knife to cut out the solidified PDMS. The cutting edge should slightly exceed the size of the chip to obtain a PDMS chip negative mold.

[0036] Step 6: Stick the processed glass sheet directly to the channel part of the PDMS chip negative mold, and ensure that there are no bubbles between the glass sheet and the PDMS chip negative mold.

[0037] Step 7: Prepare the negative mold surface treatment solution:

[0038] 1) Preparation of 0.2 mol / L Na2HPO4 solution: Dissolve 35.8 g of Na2HPO4·12H2O in 478.4 g of H2O, stir well and set aside.

[0039] 2) Preparation of 0.1 mol / L C6H8O7 solution: Dissolve 9.6 g of C6H8O7 in 500 g of H2O, stir well and set aside.

[0040] 3) Preparation of 0.3% HPMC solution by mass ratio: Dissolve 1.5 g HPMC with a viscosity of 30 mPa.s in 500 g H2O, stir well and set aside.

[0041] 4) Mix the prepared solution in a ratio of 0.2 mol / L Na2HPO4 solution: 0.1 mol / L C6H8O7 solution: 0.3% HPMC solution = 1:4:5, and stir evenly.

[0042] Step 8: Take a certain volume of negative mold surface treatment solution, immerse the PDMS chip negative mold with the glass sheet attached into the negative mold surface treatment solution, and treat for 2 hours.

[0043] Step 9: Clean the surface-treated negative mold with the glass sheet attached with deionized water and then blow dry with high-purity nitrogen.

[0044] Step 10: Place the negative mold with the glass sheet in the PC culture dish with the glass sheet facing up. Stick the PDMS directly to the bottom of the PC culture dish, exhaust the air inside, and then pour the PDMS solution into the PC culture dish.

[0045] Step 11: Place the PC culture dish in a vacuum container and evacuate the vacuum until the bubbles disappear.

[0046] Step 12: Place the vacuumed PC culture dish on a hot plate for curing at 75°C for 4 hours.

[0047] Step 13: The cured PDMS can be manually slid along the edge of the PC culture dish to remove it without cutting. Then turn the removed PDMS block upside down on the table and cut it out along the edge of the negative mold with a knife.

[0048] Step 14: Place the cut PDMS with the negative mold half facing upwards and the other half against the table. Carefully peel off the negative mold, making sure the other half is as close to the table as possible, so that the glass piece can be kept intact on the other half.

[0049] Step 15: Use a puncher to punch holes at the channel entrance and exit to obtain a PDMS microchannel cover. Step 16: Treat the PDMS microchannel cover with a PDMS coupling layer that is larger than the PDMS substrate with oxygen plasma. The side of the PDMS microchannel cover with the glass sheet is facing up. After the oxygen plasma treatment, quickly stick the two together and press with a weight for 10 minutes to complete the preparation of the glass-embedded PDMS chip with acoustic field enhancement.

Claims

1. A method for manufacturing a glass-embedded detachable surface wave acoustic fluidic sorting chip, characterized in that: The manufacturing method adopts PDMS secondary molding and demoulding of the negative mold, and the specific steps are: the first step is to prepare a PDMS negative mold by PDMS photolithography molding method, and the microchannel part in the PDMS negative mold is protruding; In the second step, the PDMS negative mold is cut and demolded according to the chip size, and a glass sheet of the corresponding size is attached to the part of the channel where the sound reflection needs to be enhanced, and then the surface is treated with the prepared surface treatment solution; In the third step, after the surface of the PDMS negative mold is cleaned, the PDMS negative mold with the glass sheet attached is placed in a container and molded with the prepared PDMS solution; in the fourth step, the whole body including the PDMS negative mold, the glass sheet and the PDMS substrate is taken out from the PC culture dish; in the fifth step, the whole body is inverted on the table for cutting, and the PDMS negative mold is demolded. The glass sheet is retained in the PDMS substrate and together with the PDMS substrate, it forms a glass-embedded PDMS substrate; Step 6: drilling holes in the glass-embedded PDMS substrate, and bonding the glass-embedded PDMS substrate and the PDMS coupling layer after plasma treatment; The microchannel is located on a glass-embedded PDMS substrate, and includes a sheath flow inlet 1, a sample inlet, a sheath flow inlet 2, a sheath flow channel 1, a sample channel, a sheath flow channel 2, a sorting channel, a glass sheet, a large cell channel, a small cell channel, a large cell outlet, and a small cell outlet; the sheath flow channel 1, the sample channel, and the sheath flow channel 2 are connected at the inlet of the sorting channel; the large cell channel and the small cell channel are connected at the outlet of the sorting channel; and the glass sheet is embedded in the top of the sorting channel.

2. The method for manufacturing a glass-embedded detachable surface wave acoustic fluidic sorting chip according to claim 1, characterized in that The surface treatment solution comprises 0.2 mol / L disodium hydrogen phosphate solution, 0.1 mol / L citric acid solution and 0.3 wt % HPMC solution in a volume ratio of 1:4:5, wherein the viscosity of HPMC is 30 mPa.s.

3. The method for manufacturing a glass-embedded detachable surface wave acoustic fluidic sorting chip according to claim 1, characterized in that: When pouring the mold, a PC flat-bottomed culture dish is used as the container, and the curing temperature is 75°C.

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