Method for producing microfluidic chips

Abstract

The application provides a production method of a micro-fluidic chip, and relates to the technical field of micro-fluidics.The micro-fluidic chip produced by the application has independent detection channels, can simultaneously detect multiple detection targets without interference, and improves the accuracy and sensitivity of detection.Meanwhile, the vacuum reagent pre-storage chamber in the detection channel of the micro-fluidic chip produced by the application comprises a reagent pre-storage chamber and a vacuum chamber, can pre-store reagents and release the reagents under vacuum negative pressure, and is particularly suitable for reagents that are not suitable for pre-freeze drying treatment, such as immunomagnetic beads or certain enzymes.

Description

[0001] This application is a divisional application of the invention application filed on October 13, 2023, with Chinese application number 202311326926.5 and title "Microfluidic Chip". Technical Field

[0002] This invention belongs to the field of microfluidics technology, and specifically relates to a method for manufacturing a microfluidic chip. Background Technology

[0003] Microfluidic chips are a current hot topic in the development of micro total analytical systems. Microfluidic chip analysis uses the chip as an operating platform, is based on analytical chemistry, relies on microelectromechanical processing (MEMS) technology, features microchannel networks as its structural characteristic, and currently focuses on life sciences as its primary application. Its goal is to integrate the entire laboratory function, including sampling, dilution, reagent addition, reaction, separation, and detection, onto a microchip.

[0004] Existing microfluidic chips typically detect only one analyte at a time. Simultaneous detection of two or more analytes requires reagent mixing, which can cause interference and affect detection results. Furthermore, current microfluidic chip manufacturing methods struggle to create multiple independent detection channels within the chip, and also make it difficult to achieve vacuum pre-storage and release of reagents within these channels. This limits the application of reagents unsuitable for pre-lyophilization, such as immunomagnetic beads or certain enzymes.

[0005] Therefore, a new method for manufacturing microfluidic chips is needed to produce microfluidic chips that can simultaneously detect multiple target objects without interference. Summary of the Invention

[0006] (a) Technical problems to be solved To address the shortcomings of existing technologies, this invention provides a method for manufacturing microfluidic chips, which solves the technical problem that existing microfluidic chips cannot simultaneously detect multiple target objects without interference.

[0007] (II) Technical Solution To achieve the above objectives, the present invention provides the following technical solution: This invention provides a method for manufacturing a microfluidic chip, comprising: Photoresist is coated on a silicon wafer, and exposure and development are performed according to the positional relationship of several detection channels to form a positive mold with several detection channels; each detection channel includes a vacuum reagent pre-storage chamber, a mixing channel, an incubation chamber and an enrichment chamber connected in sequence; the air pressure in the vacuum chamber is negative, and a thin film is provided between the reagent pre-storage chamber and the vacuum chamber; The male mold is subjected to silanization hydrophobic treatment, and an ellipsoidal polyester plastic is attached to the male mold of the incubation chamber with double-sided tape. PDMS and an initiator are poured onto the positive mold and then heated to cure, resulting in a PDMS layer with several detection channels. Pour PDMS and curing agent into an empty petri dish to obtain two patternless PDMS layers; peel the two patternless PDMS layers off the empty petri dish, cut and trim the two patternless PDMS layers after peeling, and punch holes in one of the patternless PDMS layers. Bond two unpatterned PDMS layers together; A PDMS film is bonded to the upper surface of a perforated, patternless PDMS layer; The patternless PDMS layer after bonding the PDMS film is subjected to plasma treatment. During the last plasma treatment, the PDMS film is deformed, forming a cavity at the punched location. A vacuum chamber is formed between the PDMS film and the two patternless PDMS layers. BSA was added to the concave chamber for incubation, and then the two unpatterned PDMS layers were washed with PBS to obtain the PDMS bonded layer. The PDMS layer with detection channels is peeled off from the positive mold, and then bonded to the PDMS bonding layer to obtain a microfluidic chip. During the bonding process, the groove position of the reagent pre-storage chamber in the detection channel corresponds to the drilling position.

[0008] Preferably, the positive mold further has an inlet and an outlet, the vacuum reagent pre-storage chamber is connected to the inlet, and the enrichment chamber is connected to the outlet.

[0009] Preferably, the mass ratio of PDMS to initiator is 10:1.

[0010] Preferably, the curing process involves heating at 85°C for at least 2 hours.

[0011] Preferably, plasma cleaning is performed for 1 minute before bonding, and the product is placed in an oven at 65°C for 20 minutes after bonding.

[0012] Preferably, the thickness of the PDMS film is 40-60 μm; the PDMS film is prepared by the following method: after silanizing the bare silicon wafer, the degassed PDMS and prepolymer are poured onto the silicon wafer, rotated at 1500 rpm for 60 s, and dried at 150°C for 5 min.

[0013] Preferably, the plasma treatment includes multiple plasma treatments, in which the PDMS film is deformed during the final plasma treatment.

[0014] Preferably, the BSA is a 1% BSA solution, and the incubation time is 20 min.

[0015] (III) Beneficial Effects This invention provides a method for manufacturing a microfluidic chip. Compared with the prior art, it has the following advantages: The microfluidic chip produced by this invention has independent detection channels, enabling simultaneous detection of multiple target analytes without interference, thus improving detection accuracy and sensitivity. Furthermore, the vacuum reagent pre-storage chamber in the detection channel of the microfluidic chip produced by this invention includes both a reagent pre-storage chamber and a vacuum chamber, enabling reagent pre-storage and release under negative vacuum pressure. This is particularly suitable for reagents unsuitable for pre-lyophilization, such as immunomagnetic beads or certain enzymes. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 This is a schematic diagram of the structure of a microfluidic chip according to an embodiment of the present invention; Figure 2a This is a schematic diagram of the vacuum reagent pre-storage chamber. Figure 2b A schematic diagram of the structure when the vacuum reagent pre-storage chamber is preloaded with test reagents; Figure 2c This is a schematic diagram of the structure for releasing test reagents from a vacuum reagent pre-storage chamber. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0019] This application provides a method for manufacturing a microfluidic chip, which solves the technical problem in the prior art that microfluidic chips cannot simultaneously detect multiple detection targets without interference, and enables simultaneous independent detection of multiple detection targets.

[0020] The technical solution in this application is to solve the above-mentioned technical problems, and the general idea is as follows: Existing microfluidic chips typically detect only one analyte at a time. Simultaneous detection of two or more analytes requires reagent mixing, which can cause interference and affect detection results. Furthermore, current microfluidic chip manufacturing methods struggle to create multiple independent detection channels within the chip, and also make it difficult to achieve vacuum pre-storage and release of reagents within these channels. This limits the application of reagents unsuitable for pre-lyophilization, such as immunomagnetic beads or certain enzymes.

[0021] To address the aforementioned issues, this invention proposes a method for manufacturing microfluidic chips. The microfluidic chips produced by this method have each detection channel corresponding to an independent reagent pre-storage chamber, mixing channel, incubation chamber, and enrichment chamber, allowing for the simultaneous detection of multiple target analytes without interference. The top wall of the incubation chamber is formed by an outwardly convex arc surface of ellipsoidal polyester plastic, facilitating agitation of the liquid within the chamber when combined with the vibration of a speaker diaphragm, thereby improving incubation efficiency. Simultaneously, this method allows the detection reagents to be pre-stored in the reagent pre-storage chamber under vacuum during the chip manufacturing stage. When needed, the reagents are released simply by piercing the unpatterned PDMS layer below the vacuum chamber, eliminating the need for lyophilization. This method is applicable to the production of PDMS microfluidic chips, and is particularly suitable for pre-loading reagents such as immunomagnetic beads and enzymes that are unsuitable for lyophilization.

[0022] To better understand the above technical solutions, the following will provide a detailed explanation of the technical solutions in conjunction with the accompanying drawings and specific implementation methods.

[0023] This invention provides a method for manufacturing a microfluidic chip, comprising the following steps: S1. Photoresist is coated on a silicon wafer, and exposure and development are performed according to the positional relationship of several detection channels to form a positive mold with several detection channels; each detection channel includes a vacuum reagent pre-storage chamber, a mixing channel, an incubation chamber and an enrichment chamber connected in sequence; the air pressure in the vacuum chamber is negative, and a thin film is provided between the reagent pre-storage chamber and the vacuum chamber; S2. The positive mold is subjected to silanization hydrophobic treatment, and the ellipsoidal polyester plastic is attached to the positive mold of the incubation chamber with double-sided tape. S3. Pour PDMS and initiator onto the positive mold, heat and cure to obtain a PDMS layer with several detection channels; S4. Pour PDMS and curing agent into an empty petri dish to obtain two patternless PDMS layers; peel the two patternless PDMS layers off the empty petri dish, cut and trim the two patternless PDMS layers after peeling, and punch holes in one of the patternless PDMS layers. S5. Bond the two unpatterned PDMS layers together; S6. Bond a PDMS film to the upper surface of the perforated, patternless PDMS layer; S7. Plasma treatment is performed on the patternless PDMS layer after bonding the PDMS film. During the last plasma treatment, the PDMS film deforms and forms a cavity at the punched position. A vacuum chamber is formed between the PDMS film and the two patternless PDMS layers. S8. Add BSA to the concave chamber for incubation, and then wash the two unpatterned PDMS layers with PBS to obtain the PDMS bonded layer. S9. Peel the PDMS layer with the detection channel from the positive mold, and bond the PDMS layer with the detection channel to the PDMS bonding layer to obtain a microfluidic chip. During the bonding process, the groove position of the reagent pre-storage chamber in the detection channel corresponds to the drilling position.

[0024] The microfluidic chip produced by the microfluidic chip manufacturing method of this invention includes a chip body, an inlet, an outlet, and several detection channels disposed within the chip body. Each detection channel includes a vacuum reagent pre-storage chamber, a mixing channel, an incubation chamber, and an enrichment chamber connected in sequence. The vacuum reagent pre-storage chamber is connected to the inlet, and the enrichment chamber is connected to the outlet. The sample to be tested is injected sequentially from the inlet and flows sequentially through the vacuum reagent pre-storage chamber, the mixing channel, the incubation chamber, and the enrichment chamber.

[0025] like Figure 1 As shown, the microfluidic chip includes two detection channels and has an axisymmetric structure, meaning that the two detection channels have the same structure.

[0026] The chip body of a microfluidic chip can be made of materials such as silicon, glass, and polymers. In this embodiment of the invention, a polymer is used as an example, specifically PDMS (polydimethylsiloxane).

[0027] In a specific embodiment, the microfluidic chip also includes two injection ports 10a and 10b and two liquid inlet channels 11a and 11b. One of the injection ports 10a and 10b is used to inject the sample to be tested, and the other is used to inject the washing buffer PBST. The two liquids flow into the inlet 1 through the two liquid inlet channels respectively.

[0028] The vacuum reagent pre-storage chamber includes a reagent pre-storage chamber 201, a vacuum chamber 202, and a reagent channel 203, the specific structure of which is as follows: Figure 2a As shown in the diagram, a PDMS (polydimethylsiloxane) film 204 is disposed between the reagent pre-storage chamber 201 and the vacuum chamber 202. This PDMS film has an arc surface, with an outer arc on the surface of the reagent pre-storage chamber and a patternless PDMS layer at the bottom of the vacuum chamber. A schematic diagram of the vacuum reagent pre-storage chamber when pre-loaded with detection reagents is shown below. Figure 2bAs shown; a schematic diagram of the structure when the detection reagent is released from the vacuum reagent pre-storage chamber is shown. Figure 2c As shown. In this embodiment of the invention, the thickness of the PDMS layer is 1~3 mm, for example 2 mm, and the thickness of the PDMS film is 40~60 µm, for example 50 µm. The width of the reagent channel 203 is 400~600 µm, for example 500 µm.

[0029] The reagent channel 203 of the vacuum reagent pre-storage chamber is connected to the inlet 1 via a first channel 3. The first channel 3 has a first end connected to the inlet and a second end connected to the reagent channel 203. The first channel 3 includes a wide channel section 301 and a narrow channel section 302. The wide channel section 301, connected to the inlet, has a width of 300-1000µm, for example, 500µm. The narrow channel section 302, connected to the reagent channel 203, has a width of 50-200µm, for example, 150µm. As described above, the first channel 3 includes two connecting channels of different widths. The narrow channel section increases the resistance to liquid backflow to the inlet, preventing liquid backflow to inlet 1.

[0030] The mixing channel is a serpentine mixing channel 4, with a width consistent with the wide channel segment 301, ranging from 300 to 1000 µm, for example, 500 µm. The serpentine mixing channel 4, the first channel 3, and the reagent channel 203 are connected by a T-junction. In this embodiment of the invention, the mixing channel is configured in a serpentine shape to improve the mixing effect between the sample to be tested and the detection test.

[0031] A second channel 7 is provided between the incubation chamber 5 and the enrichment chamber 6, and the width of the second channel 7 is the same as the width of the serpentine mixing channel 4. A third channel 8 is provided between the enrichment chamber 6 and the outlet 9, and the width of the third channel 8 is the same as the width of the serpentine mixing channel 4.

[0032] The microfluidic chip of this invention will be described in detail below, taking into account the fabrication and testing processes. This process is illustrated using the detection of Escherichia coli O157:H7 and Salmonella as examples.

[0033] The production process is as follows: The fabrication process of a PDMS layer with detection channels includes: Photoresist (such as SU 8-2050 photoresist) is applied to a silicon wafer, and the application and development are performed according to the positional relationship of the detection channels to form a positive mold; The cation membrane was subjected to silanization and hydrophobic treatment. An ellipsoidal polyester plastic was attached to the cation membrane of the two incubation chambers with double-sided tape. Then, PDMS and initiator (mass ratio of 10:1) were poured in and heated and cured at 85°C for more than 2 hours to obtain a PDMS layer with detection channels. The process of creating a patternless PDMS layer includes: PDMS and curing agent (mass ratio 10:1) were poured directly into an empty petri dish to obtain two patternless PDMS layers; Peel two unpatterned PDMS layers from the empty culture dish; cut and trim the two peeled PDMS layers; punch a hole in one of the unpatterned PDMS layers, since there are two reagent pre-storage chambers in this embodiment of the invention, therefore, two holes need to be punched. Two patternless PDMS layers are bonded together. It should be noted that in the embodiments of the present invention, plasma cleaning is performed for 1 min before each bonding and adhesion is promoted in an oven at 65°C for 20 min after each bonding. A PDMS film is bonded to the upper surface of a perforated, patternless PDMS layer. In this embodiment of the invention, a PDMS film with a thickness of 50µm is used. The PDMS film can be prepared by the following method: after silanizing a bare silicon wafer, degassed PDMS and prepolymer are poured onto the silicon wafer, rotated at 1500rpm for 60s, and dried at 150° for 5min.

[0034] The patternless PDMS layer after bonding the PDMS film is subjected to plasma treatment. During the last plasma treatment, the PDMS film is deformed, forming a cavity at the punched location. A vacuum chamber is formed between the PDMS film and the two patternless PDMS layers.

[0035] Add 1% BSA (bovine serum albumin) to the concave chamber and incubate for 20 minutes. Then wash the entire patternless PDMS layer after bonding with PBS (phosphate buffer) to obtain the PDMS bonded layer.

[0036] The bonding of the PDMS layer with detection channels and the PDMS bonding layer is as follows: The PDMS layer with the detection channel is peeled off from the cation membrane and then bonded to the PDMS bonding layer to obtain a microfluidic chip. During the bonding process, the groove positions of the two reagent pre-storage chambers in the detection channel correspond to the drilling positions.

[0037] It should be noted that before use, the microfluidic chip should be sealed with 1% BSA solution at room temperature for 30 minutes and rinsed with PBS to avoid non-specific adsorption of the chip.

[0038] The testing process is as follows: It should be noted that, when using it, the microfluidic chip is first attached to the speaker diaphragm; The corresponding immunoassay reagents (i.e., detection reagents) are preloaded into the reagent pre-storage chamber 202 using a capillary needle. In this embodiment of the invention, a mixture of MBs-CA1 (10 µg) and CdSe / ZnS QDMs-DA1 (15 nM) is preloaded into the left reagent pre-storage chamber, and a mixture of MBs-CA2 (10 µg) and CdSe / ZnS QDMs-DA2 (15 nM) is preloaded into the right reagent pre-storage chamber.

[0039] Bacterial solutions containing different concentrations of Escherichia coli O157:H7 and Salmonella were injected into the microfluidic chip at a flow rate of 30 µL / min. Simultaneously, the patternless PDMS layer below the two vacuum chambers was punctured with a needle to release the corresponding immunoassay reagents.

[0040] Once the solution has been mixed through the serpentine channel and reaches the incubation chamber, the injection of the bacterial solution is stopped. Music is played on a mobile phone and transmitted via Bluetooth to the speaker, causing the diaphragm to vibrate and providing agitation for the immune incubation process. After 10 minutes, the microfluidic chip is transferred to two independent external magnetic fields. Under an external magnetic field (two cylindrical magnets, each with dimensions D=4 mm, H=8 mm), the immune bacterial complex was transferred to the enrichment chamber using PBST (pH=7.4, containing 0.05% Tween-20) at a flow rate of 30 µL / min, and non-target bacteria were removed. The target bacterial complex was then enriched in the corresponding enrichment chamber. After 5 min of magnetic separation, the fluorescence signal was recorded using a smartphone and analyzed by an application.

[0041] In summary, compared with existing technologies, it has the following beneficial effects: 1. The microfluidic chip produced in the embodiments of the present invention has an independent detection channel, which can detect multiple detection targets simultaneously without interference, thereby improving the accuracy and sensitivity of detection.

[0042] 2. The vacuum reagent pre-storage chamber in the detection channel of the microfluidic chip produced in this embodiment of the invention includes a reagent pre-storage chamber and a vacuum chamber, which can realize the pre-storage and release of reagents under vacuum negative pressure, and is particularly suitable for reagents that are not suitable for pre-lyophilization, such as immunomagnetic beads or certain enzymes.

[0043] 3. The embodiments of the present invention employ PDMS soft lithography, bonding and plasma treatment processes, which can stably form PDMS layers with detection channels, patternless PDMS layers, PDMS thin films and vacuum chamber structures. The process is feasible and easy to mass-produce.

[0044] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0045] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for producing a microfluidic chip, characterized by, include: Photoresist is coated onto a silicon wafer, and exposure and development are performed according to the positional relationship of several detection channels to form a positive mold with several detection channels; Each of the detection channels includes a vacuum reagent pre-storage chamber, a mixing channel, an incubation chamber, and an enrichment chamber connected in sequence; the air pressure in the vacuum chamber is negative, and a thin film is provided between the reagent pre-storage chamber and the vacuum chamber; The male mold is subjected to silanization hydrophobic treatment, and an ellipsoidal polyester plastic is attached to the male mold of the incubation chamber with double-sided tape. PDMS and an initiator are poured onto the positive mold and then heated to cure, resulting in a PDMS layer with several detection channels. Pour PDMS and curing agent into an empty petri dish to obtain two patternless PDMS layers; peel the two patternless PDMS layers off the empty petri dish, cut and trim the two patternless PDMS layers after peeling, and punch holes in one of the patternless PDMS layers. Bond two unpatterned PDMS layers together; A PDMS film is bonded to the upper surface of a perforated, patternless PDMS layer; Plasma treatment was performed on the patternless PDMS layer after bonding the PDMS film. During the last plasma treatment, the PDMS film deformed and formed a cavity at the punched location. A vacuum chamber was formed between the PDMS film and the two patternless PDMS layers. BSA was added to the concave chamber for incubation, and then the two unpatterned PDMS layers were washed with PBS to obtain the PDMS bonded layer. The PDMS layer with detection channels is peeled off from the positive mold, and then bonded to the PDMS bonding layer to obtain a microfluidic chip. During the bonding process, the groove position of the reagent pre-storage chamber in the detection channel corresponds to the drilling position.

2. The method of producing a microfluidic chip according to claim 1, wherein The positive mold also has an inlet and an outlet, the vacuum reagent pre-storage chamber is connected to the inlet, and the enrichment chamber is connected to the outlet.

3. The method of producing a microfluidic chip according to claim 1, wherein The mass ratio of PDMS to initiator is 10:

1.

4. The method of producing a microfluidic chip according to claim 1, wherein The heat curing includes heating and curing at 85°C for more than 2 hours.

5. The method of producing a microfluidic chip according to claim 1, wherein The production method further includes: performing plasma cleaning for 1 minute before bonding, and placing the product in a 65°C oven for 20 minutes after bonding.

6. The method of producing a microfluidic chip according to claim 1, wherein The thickness of the PDMS film is 40-60 μm; the PDMS film is prepared by the following method: after silanizing the bare silicon wafer, the degassed PDMS and prepolymer are poured onto the silicon wafer, rotated at 1500 rpm for 60 s, and dried at 150°C for 5 min.

7. The method of producing a microfluidic chip according to claim 1, wherein The plasma treatment includes multiple plasma treatments, with the PDMS film deformed during the final plasma treatment.

8. The method of producing a microfluidic chip according to claim 1, wherein The BSA is a 1% BSA solution, and the incubation time is 20 min.

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