Microfluidic chip device, preparation method and microsphere loading method

Abstract

The application provides a microfluidic chip device, a preparation method and a microsphere loading method, and relates to the technical field of microfluidic chips. The microfluidic chip device comprises a chip, and the chip comprises: a first layer, a microhole array is arranged in the middle of the upper part of the first layer, 200000-300000 microholes are arranged in the microhole array, and the top of the microholes is open; and a second layer, which is arranged above the first layer and is bonded to the first layer, a flow channel is arranged in the lower part of the second layer, an inlet and an outlet are arranged at the two ends of the flow channel, a detection area is arranged in the middle of the flow channel, the inlet, the outlet and the detection area are in communication with the flow channel, and the bottom of the detection area is open and covers the microhole array. The microspheres in the microhole array of the microfluidic chip device provided by the application are in a monodisperse state and cannot agglomerate, and the loading rate of the microspheres in the entire microhole array reaches more than 90%.

Description

Technical Field

[0001] This invention relates to the field of microfluidic chip technology, specifically to a microfluidic chip device, a fabrication method, and a microsphere loading method. Background Technology

[0002] With the development of science and technology, the continuous progress of medicine, and the improvement of life analysis instruments, scientists' exploration of the field of life analysis has gradually reached new heights, and higher requirements have been placed on the detection of biomarkers. In order to explore the heterogeneity between molecules and cells, traditional detection systems that collect average signals from a large number of molecules are insufficient to meet practical needs. Digital analysis, on the other hand, can explore the information of individual molecules, and under certain conditions, even achieve absolute quantitative analysis. This provides a powerful quantitative method for the detection of biomarkers in biomedical research, with enormous potential practical value and high commercial prospects.

[0003] Digital analysis relies on the preparation of large quantities of micro / nano-scale reaction supports. These supports need to be uniform in size and regularly arranged. Directly using a large number of magnetic microspheres in the reaction and then dropping them onto a glass slide for detection will cause the magnetic microspheres to aggregate rather than be monodisperse, making them unsuitable for digital analysis. Most existing methods for preparing digital microarray analysis platforms are only applicable to one-pot reactions and require relatively expensive specialized instruments.

[0004] Therefore, there is a need to study a microfluidic chip device that can uniformly disperse and clean magnetic microspheres in order to prepare microarray substrates and provide better reaction carriers for digital analysis. Summary of the Invention

[0005] Based on the above analysis, the present invention aims to provide a microfluidic chip device, a fabrication method, and a microsphere loading method to solve at least one of the following problems: enabling microspheres to be uniformly dispersed without agglomeration, and enabling microspheres to be cleaned in subsequent analytical operations.

[0006] The objective of this invention is mainly achieved through the following technical solutions:

[0007] In a first aspect, the present invention provides a microfluidic chip device, comprising a chip, the chip comprising: a first layer, wherein a micropore array is disposed in the middle of the upper part of the first layer, wherein 200,000-300,000 micropores are disposed in the micropore array, and the top of the micropores are open; and a second layer, disposed above the first layer and bonded to the first layer, wherein a flow channel is disposed in the lower part of the second layer, wherein an inlet and an outlet are disposed at both ends of the flow channel, and a detection area is disposed in the middle of the flow channel, wherein the inlet, the outlet, and the detection area are all connected to the flow channel, and the bottom of the detection area is open and covers the micropore array.

[0008] Preferably, the microfluidic chip device further includes a glass slide disposed below the first layer, and the glass slide is bonded to the first layer.

[0009] Preferably, the microfluidic chip device further includes a magnet support disposed below the chip and detachably connected to the chip.

[0010] Preferably, the diameters of the inlet and the outlet are 1-3 mm, the length of the flow channel on either side of the detection area is 4-6 mm, the width and height of the flow channel are both 100-300 micrometers, the diameter of the detection area is 4-6 mm, the height of the detection area is 100-300 micrometers, the diameter of the micropore array is 3-5 mm, the diameter of the micropore is 4-6 micrometers, the depth of the micropore is 3-5 micrometers, and the spacing between adjacent micropores is 2-4 micrometers.

[0011] Secondly, the present invention provides a method for fabricating a microfluidic chip device, comprising the following steps: S1: fabricating a second layer; S2: fabricating a first layer; and S3: bonding the second layer to the first layer by an oxygen plasma bonding method, wherein the detection area of ​​the second layer is aligned with the micropore array of the first layer to obtain the chip.

[0012] Preferably, step S1 includes the following steps: S1-1: Under the protection of a mask, SU8-3050 photoresist is used to etch onto a single-crystal silicon wafer, and a mold for casting is prepared by wet photolithography; S1-2: Dimethylsiloxane and initiator are mixed at a mass ratio of (9-11):1, and after vacuuming to remove air bubbles, the mixture is poured into the mold prepared in step S1-1; and S1-3: After vacuuming again to remove invisible air bubbles, the mixture is heated and cured. After removing the cured polydimethylsiloxane, holes are drilled at the inlet and outlet to obtain the second layer.

[0013] Preferably, step S2 includes the following steps: S2-1: Under the protection of a mask, SU8-3050 photoresist is used to etch onto a single-crystal silicon wafer, and a mold for casting is prepared by wet photolithography; S2-2: Dimethylsiloxane and initiator are mixed at a mass ratio of (9-11):1, and after vacuuming to remove air bubbles, the mixture is poured into the mold prepared in step S2-1; and S2-3: After vacuuming again to remove air bubbles that are not visible to the naked eye, the mixture is heated to cure, and the cured polydimethylsiloxane is taken out to obtain the first layer.

[0014] Preferably, the method further includes the following step: S4: bonding the chip to the glass slide using an oxygen plasma bonding method.

[0015] Thirdly, the present invention provides a microsphere loading method using the aforementioned microfluidic chip device, comprising the following steps: Step 1: injecting a solution containing magnetic microspheres into the inlet of the chip, wherein the micropore array region is filled with the magnetic microspheres; Step 2: after settling, the magnetic microspheres enter the micropores by gravity; and Step 3: rinsing the magnetic microspheres not in the micropores on the chip at a rate of 0.05-0.2 μL / min two or more times, each rinsing time being 30-120 min.

[0016] Preferably, the microsphere loading method further includes step 4: placing the chip loaded with the magnetic microspheres obtained in step 3 onto the magnet support, so that the magnetic microspheres are magnetically connected to the magnet support, allowing the microfluidic chip device to be used for subsequent analysis; and / or the concentration of the magnetic microspheres in the solution containing the magnetic microspheres is 4.8 × 10⁻⁶. 4 From 1.2 × 10⁻⁶ cells / μL to 1.2 × 10⁻ 5 The number of magnetic microspheres in the solution containing magnetic microspheres is 1-3 times the number of micropores.

[0017] Compared with the prior art, the present invention can achieve at least one of the following beneficial effects:

[0018] A) The microfluidic chip device provided by this invention allows a solution containing microspheres to flow into a micropore array in the detection area through an inlet and a flow channel. The microspheres enter the micropores in the micropore array and are in a monodisperse state, preventing aggregation. This allows for digital analysis. Furthermore, the microfluidic chip device provided by this invention can clean the microspheres in the micropore array. The cleaning liquid flows in from the inlet, passes through the micropore array, and flows out from the outlet, making the application of the microfluidic chip more extensive.

[0019] B) The microsphere loading method provided by the present invention optimizes the concentration and flow rate of the injected magnetic microspheres, thereby enabling each micropore to load only one microsphere, and the loading rate of the microspheres in the entire micropore array reaches more than 90%.

[0020] C) The microsphere loading method provided by the present invention, after the microspheres are loaded, the chip and glass slide are placed on the magnetic support as the microarray substrate, which further ensures that the microspheres in the holes will not be flushed out of the micropores after multiple reactions and cleanings in subsequent analysis experiments.

[0021] C) The microfluidic chip device provided by this invention is expected to provide a new reaction platform for digital analysis of subsequent multi-step reactions, and is applicable to a wider range of absolute quantitative analysis methods for biomarkers. Attached Figure Description

[0022] Figure 1 This is a perspective view of the microfluidic chip device provided by the present invention.

[0023] Figure 2 This is a schematic diagram of the structure of the microfluidic chip device provided by the present invention.

[0024] Figure 3 This is a schematic diagram of the micropore array of the microfluidic chip device provided by the present invention.

[0025] Figure 4 This is a schematic diagram of the micropore array before and after the microspheres are inserted into the pores.

[0026] Figure 5 These are bright-field microscopic images of the microspheres before insertion into the well, before insertion without cleaning, and after cleaning.

[0027] Figure 6 This is a physical image of the microfluidic chip device provided by the present invention.

[0028] Figure 7 Bright-field micrographs of the micropore array before and after cleaning, after injecting different numbers of microspheres into the chip.

[0029] Explanation of reference numerals in the attached figures:

[0030] 100-Chip; 110-First layer; 111-Microwell array; 112-Microwell; 120-Second layer; 121-Flow channel; 122-Inlet; 123-Outlet; 124-Detection area; 200-Slide; 300-Magnet support. Detailed Implementation

[0031] Hereinafter, embodiments thereof, along with their various features and advantageous details, are explained more fully with reference to non-limiting embodiments illustrated in the accompanying drawings and described in detail in the following description. Descriptions of well-known components and processing techniques have been omitted to avoid unnecessarily obscuring the embodiments herein. Furthermore, the various embodiments described herein are not necessarily mutually exclusive, as some embodiments may be combined with one or more other embodiments to form new embodiments. Unless otherwise stated, the term "or" as used herein means non-exclusive or. The examples used herein are intended only to facilitate an understanding of how the embodiments herein can be practiced and to further enable those skilled in the art to practice the embodiments herein. Therefore, the examples should not be construed as limiting the scope of the embodiments herein.

[0032] In the description of this invention, it should be understood that the terms "center," "longitudinal," "lateral," "length," "width," "thickness," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," "clockwise," "counterclockwise," "axial," "radial," and "circumferential" indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are used only for the convenience of describing this invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this invention.

[0033] Traditional biomarker detection methods typically obtain biomarker information by acquiring the average simulated signal (e.g., total fluorescence intensity) generated by a large number of biomarkers. Relative quantitative analysis of biomarkers is then indirectly achieved by establishing a calibration curve between the simulated signal generated by standards and the concentration of the target biomarker. Unlike traditional methods, digital quantitative analysis offers unique advantages. It eliminates the need for calibration curves to quantify biomarkers in samples, enabling absolute quantitative analysis. In digital quantitative analysis, the reaction system is divided into numerous microreactors. The target biomarker molecule is diluted to a specific concentration, and through certain techniques, most reactors contain at most one copy of the target molecule. After the reaction, microreactors loaded with one or zero target molecules output "1" or "0," respectively. Only microreactors displaying positive signals are counted among the numerous independent microreactors; the overall signal intensity of the reaction system is not statistically analyzed. By counting the number of positive microreactors and combining this with a Poisson distribution, absolute quantitative analysis of the target molecule can be achieved.

[0034] In recent years, thanks to the unremitting efforts of researchers, a number of commercial instruments based on digital absolute quantitative analysis of biomarkers have emerged, among which the most widely used are droplet digital PCR technology (ddPCR) and single-molecule array technology (Simoa).

[0035] Digital PCR technology generates thousands of water-in-oil droplets using a droplet generator. Each droplet contains at most one copy of the target DNA molecule, and the target DNA molecules are distributed according to a Poisson distribution. Each droplet then acts as a microreactor for PCR amplification. After amplification, each droplet is individually tested. Only droplets containing the target molecule produce a fluorescent signal and are output as "1". Droplets without the target molecule do not produce a fluorescent signal and are output as "0". By counting all droplets, the number of positive and negative droplets can be obtained. Based on the Poisson distribution, the initial copy number or initial concentration of the target nucleic acid molecule can be determined.

[0036] Simoa single-molecule array technology immobilizes and captures antibodies on microspheres, which then undergo a sandwich immunoreaction with antigens and biotin-labeled detection antibodies. When the antigen concentration in the solution is extremely low, the ratio of antigen to microspheres is less than 1:1. The proportion of microspheres containing the sandwich immune complex follows a Poisson distribution, with each microsphere containing either one or zero immune complexes. The addition of STV-modified galactosidase binds to the biotin, resulting in one or zero galactosidase molecules on each microsphere. The microspheres are then added into micropores on the surface of an optical fiber bundle. By controlling the size of the micropores, each micropore contains only one microsphere. A special oil is then used for sealing. Because a single enzyme is confined within a micropore, high fluorescence intensity is ensured locally in each micropore. Fluorescence imaging of the micropore array and counting of positive micropores are then used for immunoassay, enabling digital ultra-high sensitivity detection of antigens.

[0037] However, both the microdroplets used in ddPCR and the micropore arrays on the fiber bundle surface used in Simoa technology are only suitable for one-pot reactions, and cannot be cleaned after sealing. ddPCR requires specific and relatively expensive dedicated instruments (ddRCR instruments). The steps of etching micropores on the fiber bundle surface, loading microspheres by centrifugation, and sealing are all quite cumbersome. Therefore, establishing an integrated, easy-to-clean, and inexpensive digital reaction device remains a significant challenge.

[0038] Firstly, referring to Figure 1-4 The present invention provides a microfluidic chip device, including a chip 100, wherein the chip 100 includes:

[0039] The first layer 110 has a micropore array 111 in the middle of the upper part of the first layer 110. The micropore array 111 has 200,000-300,000 micropores 112, and the top of the micropores 112 is open.

[0040] The second layer 120 is disposed above the first layer 110 and is bonded to the first layer 110. A flow channel 121 is disposed at the lower part of the second layer 120. An inlet 122 and an outlet 123 are disposed at both ends of the flow channel 121. A detection area 124 is disposed in the middle of the flow channel 121. The inlet 122, the outlet 123, and the detection area 124 are all connected to the flow channel 121. The bottom of the detection area 124 is open and covers the micropore array 111.

[0041] It should be noted that the microfluidic chip device provided by the present invention allows a solution containing microspheres to flow into a micropore array in the detection area through an inlet and a flow channel. The microspheres enter the micropores in the micropore array and are in a monodisperse state, preventing aggregation. This allows for digital analysis. Furthermore, the microfluidic chip device provided by the present invention can clean the microspheres in the micropore array. The cleaning liquid flows in from the inlet, passes through the micropore array, and flows out from the outlet, making the application of the microfluidic chip more extensive.

[0042] In one specific embodiment of the present invention, the microfluidic chip device further includes a glass slide 200 disposed below the first layer 110. It should be noted that the first layer 110, the second layer 120, and the glass slide 200 of the chip 100 can be connected by oxygen plasma bonding.

[0043] As a specific embodiment of the present invention, the microfluidic chip device provided by the present invention further includes a magnet holder 300, and the chip 100 is detachably connected to the magnet holder 300. The magnet holder 300 can be a container or device containing magnets, for example, the magnets can be placed in a housing or container with a receiving cavity. In some cases, the magnets may or may not be in contact with the glass slide 200, depending on whether the magnetic microspheres in the chip 100 are magnetically connected to the magnets.

[0044] In one specific embodiment of the present invention, the flow channel 121 can be trough-shaped and open at the bottom of the second layer 120. In another specific embodiment of the present invention, the flow channel 121 can also be closed at the bottom and configured as an internal channel. Similarly, the inlet 122 and the outlet 123 can be open at the bottom of the second layer 120 or closed, provided that the flow channel 121, the inlet 122, and the outlet 123 allow fluid to enter the detection area 124 and then the micropore array 111.

[0045] In one specific embodiment of the present invention, the diameters of the inlet 122 and the outlet 123 can be 1-3 mm, preferably 2 mm; the length of the flow channel 121 on either side of the detection area 124 is 4-6 mm, preferably 5 mm; the width and height of the flow channel 121 can both be 100-300 micrometers, preferably 200 micrometers; the detection area 124 in the middle can be circular, with a diameter of 4-6 mm, preferably 5 mm; the height of the detection area 124 can be 100-300 micrometers, preferably 200 micrometers.

[0046] In one specific embodiment of the present invention, the micropore array 111 can be circular with a diameter of 3-5 mm, preferably 4 mm; the micropores 112 can be cylindrical with a diameter of 4-6 micrometers, preferably 4.5 micrometers; the depth of the micropores 112 can be 3-5 micrometers, preferably 3.8 micrometers; the spacing between adjacent micropores 112 can be 2-4 micrometers, preferably 3 micrometers; and the number of micropores can be 200,000-300,000, preferably 222,211.

[0047] Secondly, the present invention provides a method for fabricating a microfluidic chip device, comprising the following steps:

[0048] S1: Preparation of the second layer 120;

[0049] S2: Preparation of the first layer 110;

[0050] S3: The second layer 120 is bonded to the first layer 110 by oxygen plasma bonding method, and the detection area 124 of the second layer 120 is aligned with the micropore array 111 of the first layer 110.

[0051] S4: Bond chip 100 to glass slide 200 using oxygen plasma bonding.

[0052] As a specific embodiment of the present invention, step S1 includes the following steps:

[0053] S1-1: Under the protection of a photomask, SU8-3050 photoresist is used to etch the mold onto a single-crystal silicon wafer, and wet photolithography is used to prepare the mold for casting.

[0054] S1-2: Mix dimethylsiloxane (DMS) and its initiator at a ratio of (9-11):1 (w:w), remove air bubbles by vacuuming, and then pour the mixture into the mold described above.

[0055] S1-3: After removing invisible air bubbles by vacuuming again, heat and cure. After removing the cured polydimethylsiloxane (PDMS), use a punch to punch holes in inlet 122 and outlet 123 respectively to obtain the second layer 120.

[0056] As a specific embodiment of the present invention, the initiator can be SYLGARD184 silicone rubber.

[0057] As a specific embodiment of the present invention, step S2 includes the following steps:

[0058] S2-1: Under the protection of a photomask, SU8-3050 photoresist is used to etch the mold onto a single-crystal silicon wafer, and wet photolithography is used to prepare the mold for casting.

[0059] S2-2: Mix dimethylsiloxane (DMS) and its initiator at a ratio of (9-11):1 (w:w), remove air bubbles by vacuuming, and then pour the mixture into the mold described above.

[0060] S2-3: After removing invisible air bubbles by vacuuming again, heat to cure. Remove the cured polydimethylsiloxane (PDMS) from the mold to obtain the first layer 110.

[0061] As a specific embodiment of the present invention, the method for fabricating the microfluidic chip device provided by the present invention further includes the following steps:

[0062] S5: Preparation of the magnet support: The preparation process is as follows: The design drawing of the magnet support is drawn using AutoCAD software. Then, the 2 mm thick polymethyl methacrylate (PMMA) board is cut into the required shape according to the design drawing using a laser cutting machine. Finally, the PMMA slices are assembled into the required support using dichloromethane, and the magnet is fixed on the support.

[0063] As a specific embodiment of the present invention, the specific operation of step S1-1 is as follows: Take a single crystal silicon wafer, heat it at 135°C overnight, cool it, activate it with a plasma cleaner, then pour about 5g of SU8-3050 photoresist and spin coat it (spray coating parameters: 100 rpm, 10 s; 2350 rpm, 55 s; 100 rpm, 10 s); then heat it at 65°C for 1 min, then transfer it to 95°C for 20 min to evaporate the solvent in the photoresist; after cooling, expose it with a photolithography machine (exposure energy: 130mJ / cm). 2 After exposure and baking (heating at 65°C for 1 minute, then transferring to 95°C for 6 minutes), the uncrosslinked photoresist is washed away with developer to obtain a chip mold that meets the design requirements.

[0064] As a specific embodiment of the present invention, the specific operation of step S2-1 is as follows: Take a single crystal silicon wafer, heat it at 135°C overnight, cool it, activate it with a plasma cleaner, then pour about 5g of SU8-3050 photoresist and spin coat it (spray coating parameters: 1000rpm, 20s; 20000rpm, 360s; 1000rpm, 20s); then heat it at 65°C for 1min, and then transfer it to 95°C for 20min to evaporate the solvent in the photoresist; after cooling, expose it with a photolithography machine (exposure energy: 130mJ / cm). 2 After exposure and baking (heating at 65°C for 1 minute, then transferring to 95°C for 6 minutes), the uncrosslinked photoresist is washed away with developer to obtain a chip mold that meets the design requirements.

[0065] Thirdly, the present invention provides a microsphere loading method, comprising the following steps:

[0066] Step 1: The solution containing magnetic microspheres is injected through the inlet 122 of the chip 100, and the micropore array 111 is filled with microspheres;

[0067] Step 2: After standing for 20-30 minutes, the microspheres enter the micropores by gravity;

[0068] Step 3: Rinse the microspheres on the chip that are not in the holes twice at a rate of 0.05-0.2 μL / min, each rinse lasting 30-120 min;

[0069] Step 4: Place the chip 100 loaded with microspheres onto the magnet holder, so that the microspheres are magnetically connected to the magnet holder, so that the microfluidic chip device can be used for subsequent analysis.

[0070] In one embodiment of the present invention, the concentration of the microspheres in the solution containing the magnetic microspheres is 4.8 × 10⁻⁶. 4 From 1.2 × 10⁻⁶ cells / μL to 1.2 × 10⁻ 5 The number of injected magnetic microspheres is 1-3 times the number of micropores per μL.

[0071] As one embodiment of the present invention, the solution containing magnetic microspheres can be made by dispersing the magnetic microspheres in a 1×PBST solution (1×PBS contains 0.1% Tween-20), wherein the 1×PBS is 10 mM, pH 7.4, containing 137 mM NaCl and 2.7 mM KCl.

[0072] As one embodiment of the present invention, the microspheres are rinsed with deionized water.

[0073] Figure 5 Image (a) is a bright-field micrograph of the micropore array before the microspheres are inserted into the pores. Figure 5 Image (b) is a bright-field micrograph after microsphere injection but before excess microspheres are cleaned. Figure 5 Image (c) shows a bright-field micrograph of the microporous array of injected microspheres after cleaning. Figure 5 It can be seen that at most one microsphere can enter each micropore. The cleaning process only washes away the microspheres that have not entered the pore, without causing the microspheres to overflow from the pore.

[0074] The microsphere loading method provided by this invention optimizes the concentration and flow rate of injected magnetic microspheres, achieving a loading rate of over 90% for each microsphere in the entire microwell array by loading only one microsphere per microwell. After loading, the chip and glass slide serve as the microarray substrate, placed on a magnet-equipped support to further ensure that the microspheres in the wells are not flushed out after multiple reactions and cleanings in subsequent analytical experiments. This invention provides a microfluidic chip device that promises to offer a new reaction platform for subsequent multi-step digital analysis, applicable to a wider range of absolute quantitative analysis methods for biomarkers.

[0075] The following detailed description of preferred embodiments of the present invention illustrates the principles of the invention and is not intended to limit the scope of the invention.

[0076] Example 1

[0077] Chip fabrication

[0078] S1: Preparation of the second layer 120; the specific steps of S1 are as follows:

[0079] S1-1: Under the protection of a photomask, SU8-3050 photoresist is used to etch the image onto a single-crystal silicon wafer. Wet photolithography is then used to fabricate the mold for casting. The specific steps are as follows:

[0080] The design of the second layer 120 of the chip was drawn using AutoCAD software. The diameters of the inlet 122 and outlet 123 of the flow channel 121 are both 2 mm. The length of the flow channel 121 on either side of the detection area 124 is 5 mm. The width and height of the flow channel 121 are both 200 micrometers. The diameter of the central circular detection area 124 is 5 mm and the height is 200 micrometers. A chromium plate mask was fabricated based on the design of the second layer 120 of the chip.

[0081] The die used for chip casting employs wet photolithography, with SU8-3050 photoresist applied to a single-crystal silicon wafer. The specific procedure is as follows: The single-crystal silicon wafer is heated to 135°C overnight. After cooling, it is activated using a plasma cleaner. Approximately 5g of SU8-3050 photoresist is then poured onto a spin coater (spray coating parameters: 100rpm, 10s; 2350rpm, 55s; 100rpm, 10s). Subsequently, it is heated to 65°C for 1 minute, then transferred to 95°C for 20 minutes to evaporate the solvent from the photoresist. After cooling, it is exposed using a photolithography machine (exposure energy: 130 mJ / cm²). 2 After exposure and baking (heating at 65℃ for 1 minute, then transferring to 95℃ for 6 minutes), and cooling, the uncrosslinked photoresist is washed away with developer to obtain a chip mold that meets the design requirements.

[0082] S1-2: Mix dimethylsiloxane (DMS) and its initiator at a ratio of 10:1 (w:w), remove air bubbles by vacuuming, and pour into the mold mentioned above.

[0083] S1-3: After removing invisible air bubbles by vacuuming again, heat at 95℃ for 30 min to cure. After removing the cured PDMS, use a punch to drill holes at the inlet and outlet respectively.

[0084] S2: Preparation of the first layer 110; the specific steps of S2 are as follows:

[0085] S2-1: Under the protection of a photomask, SU8-3050 photoresist is used to etch the image onto a single-crystal silicon wafer. Wet photolithography is then used to fabricate the mold for casting. The specific steps are as follows:

[0086] The design of the first layer 110 of the chip was drawn using AutoCAD software. The diameter of the micro-hole array 111 is 4 mm, the diameter of the micro-hole 112 is 4.5 micrometers, the height is 3.8 micrometers, the spacing between adjacent micro-holes 112 is 3 micrometers, and the number of micro-holes is 222211. A chromium plate mask was prepared according to the design of the upper layer of the chip.

[0087] The die used for chip casting employs wet photolithography, with SU8-3050 photoresist applied to a single-crystal silicon wafer. The specific procedure is as follows: The single-crystal silicon wafer is heated to 135°C overnight. After cooling, it is activated using a plasma cleaner. Approximately 5g of SU8-3050 photoresist is then poured and spin-coated (spray coating parameters: 1000rpm, 20s; 20000rpm, 360s; 1000rpm, 20s). Subsequently, it is heated to 65°C for 1 minute, then transferred to 95°C for 20 minutes to evaporate the solvent from the photoresist. After cooling, it is exposed using a photolithography machine (exposure energy: 130mJ / cm²). 2 After exposure and baking (heating at 65℃ for 1 minute, then transferring to 95℃ for 6 minutes), and cooling, the uncrosslinked photoresist is washed away with developer to obtain a chip mold that meets the design requirements.

[0088] S2-2: Mix dimethylsiloxane (DMS) and its initiator at a ratio of 10:1 (w:w), remove air bubbles by vacuuming, and pour into the mold mentioned above.

[0089] S2-3: After removing invisible air bubbles by vacuuming again, heat at 95℃ for 30 min to cure. Remove the cured PDMS from the mold to obtain the first layer 110.

[0090] S3: The assembly method of the second layer 120 and the first layer 110 of the chip 100 includes:

[0091] The second layer 120 is bonded to the first layer 110 using oxygen plasma bonding, and the middle detection region 124 of the second layer 120 is aligned with the micropore array 111 of the first layer 110.

[0092] S4: Bond chip 100 to the glass slide using oxygen plasma bonding.

[0093] S5: Fabrication of the magnet support:

[0094] The design of the magnet bracket was drawn using AutoCAD software. Then, a 2mm thick polymethyl methacrylate (PMMA) sheet was cut into the required shape using a laser cutter according to the design. Finally, the PMMA slices were assembled into the required bracket using dichloromethane, and the magnet was fixed on the bracket.

[0095] Example 2

[0096] The microsphere loading method, using the chip prepared in Example 1, includes the following steps:

[0097] Step 1: Add 5 μL of a solution with a concentration of 1.2 × 10⁻⁶ 5 microspheres / μL (6×10⁶ microspheres) 5 Magnetic microspheres (DynabeadsTMM-270 Streptavidin, Invitrogen) (approximately 2.7 times the number of micropores) are injected through inlet 122 of chip 100, and the 4 mm micropore array 111 in the middle is filled with microspheres.

[0098] Step 2: After standing for 20 minutes, allow the microspheres to enter the micropores by gravity;

[0099] Step 3: Rinse the microspheres on the chip that are not in the holes twice at a rate of 0.1 μL / min, 60 min each time;

[0100] Step 4: Place the chip loaded with microspheres onto the magnet support so that the microfluidic chip device can be used for subsequent analysis.

[0101] Example 3

[0102] The microsphere loading method, using the chip prepared in Example 1, includes the following steps:

[0103] Step 1: Add 5 μL of a solution with a concentration of 4.8 × 10⁻⁶ 4 microspheres / μL (2.4 × 10⁻⁶) 5 Magnetic microspheres (DynabeadsTMM-270 Streptavidin, Invitrogen) (equivalent to 1.08 times the number of micropores) are injected through the chip inlet, and the central 4 mm micropore array 111 is filled with microspheres;

[0104] Step 2: After standing for 20 minutes, allow the microspheres to enter the micropores by gravity;

[0105] Step 3: Rinse the microspheres on the chip that are not in the holes twice at a rate of 0.1 μL / min, 60 min each time;

[0106] Step 4: Place the chip loaded with microspheres onto the magnet support so that the microfluidic chip device can be used for subsequent analysis.

[0107] Comparative Example 1

[0108] This comparative example is basically the same as Example 2, except that the concentration of the microsphere solution is 2.4 × 10⁻⁶. 4 cells / μL (1.2×10⁻⁶) 5 Each microsphere is approximately 0.54 times the number of micropores.

[0109] Comparative Example 2

[0110] This comparative example is basically the same as Example 2, except that the concentration of the microsphere solution is 2.4 × 10⁻⁶. 5 cells / μL (1.2×10⁻⁶) 6 Each microsphere is approximately 5.4 times the number of micropores.

[0111] Experimental Example 1

[0112] For the microfluidic chip device prepared in Example 1, the fluid flow in the microfluidic chip device was tested using dye as the flow medium. Figure 6 This is a physical image of the microfluidic chip device provided by the present invention. Figure 6 (a) is a front view of the chip after the second layer 120 and the first layer 110 are bonded to the glass slide 200. Figure 6 (b) After the dye is injected into the chip, the flow channel is unobstructed and the central circular detection area 124 can be filled with solution. Figure 6 (c) in the image is a front view of the chip placed on the magnet holder. Figure 6 (d) is a front view of the dye-injected chip placed on a magnet holder. Figure 6 (e) in the image is a side view of the chip placed on the magnet holder. Figure 6 (f) is a side view of the dye-injected chip placed on a magnet holder.

[0113] Experimental Example 2

[0114] Adding microspheres of different concentrations or quantities to the chip results in varying microsphere penetration rates in the microporous array. For the microsphere loading methods of Examples 2-3 and Comparative Examples 1-2, the chip was observed using a bright-field microscopy microscope before being placed onto the magnet support in step 4.

[0115] like Figure 7As shown in (c) and (d) in Example 2, 5 μL of a solution with a concentration of 1.2 × 10⁻⁶ was injected. 5 microspheres / μL (6×10⁶ microspheres) 5 The solution contains magnetic microspheres (approximately 2.7 times the number of micropores), and microspheres that are not in the pores are easily washed away (step 3), with a microsphere penetration rate as high as 99.8%.

[0116] like Figure 7 As shown in (e) and (f) in Example 3, 5 μL of the solution was injected at a concentration of 4.8 × 10⁻⁶. 4 microspheres / μL (2.4 × 10⁻⁶) 5 A solution of magnetic microspheres (equivalent to 1.08 times the number of micropores) was prepared, and the microspheres that did not enter the pores were easily washed away (step 3), with a microsphere penetration rate of 92%.

[0117] like Figure 7 As shown in (g) and (h), Comparative Example 1 was injected with 5 μL of a concentration of 2.4 × 10⁻⁶. 4 cells / μL (1.2×10⁻⁶) 5 The magnetic microsphere solution contains only 74.8% microspheres (approximately 0.54 times the number of micropores). The number of microspheres is too small, resulting in a microsphere penetration rate of only 74.8%.

[0118] like Figure 7 As shown in (a) and (b) in the figures, Comparative Example 2 was injected with 5 μL of a concentration of 2.4 × 10⁻⁶. 5 cells / μL (1.2×10⁻⁶) 6 A magnetic microsphere solution containing 100 microspheres (approximately 5.4 times the number of micropores) is difficult to clean off due to the large number of microspheres, and microspheres that are not in the pores are prone to clogging the flow channels.

[0119] Any numerical value mentioned in this invention, if there is only a two-unit interval between any minimum and any maximum value, includes all values ​​that increase by one unit each time from the minimum to the maximum value. For example, if the amount of a component, or the value of a process variable such as temperature, pressure, or time, is stated as 50-90, in this specification it means specifically listing values ​​such as 51-89, 52-88… and 69-71 and 70-71, etc. For non-integer values, it may be appropriately considered that a unit is 0.1, 0.01, 0.001, or 0.0001. These are merely some specifically specified examples. In this application, in a similar manner, all possible combinations of numerical values ​​between the listed minimum and maximum values ​​are considered to have been disclosed.

[0120] It should be noted that the embodiments described above are only for explaining the present invention and do not constitute any limitation on the present invention. The present invention has been described with reference to typical embodiments, but it should be understood that the words used therein are descriptive and explanatory terms, not limiting terms. Modifications can be made to the present invention within the scope of the claims, and revisions can be made to the present invention without departing from the scope and spirit of the present invention. Although the present invention described herein relates to specific methods, materials, and embodiments, it does not mean that the present invention is limited to the specific examples disclosed herein; on the contrary, the present invention can be extended to all other methods and applications with the same function.

Claims

1. A microsphere loading method, employing a microfluidic chip device, wherein the microfluidic chip device comprises a chip, characterized in that, The chip includes: The first layer has a micropore array in the upper middle part, wherein the micropore array contains 200,000-300,000 micropores, and the top of each micropore is open; and The second layer is disposed above the first layer and bonded to the first layer. A flow channel is provided at the bottom of the second layer. An inlet and an outlet are provided at both ends of the flow channel. A detection area is provided in the middle of the flow channel. The inlet, the outlet, and the detection area are all connected to the flow channel. The bottom of the detection area is open and covers the micropore array. The microsphere loading method includes the following steps: Step 1: A solution containing magnetic microspheres is injected into the inlet of the chip, and the micropore array region is filled with the magnetic microspheres; Step 2: After settling, the magnetic microspheres enter the micropores by gravity; and Step 3: Rinse the magnetic microspheres on the chip that are not in the holes two or more times at a rate of 0.05-0.2 μL / min, with each rinsing time being 30-120 min; The concentration of the magnetic microspheres in the solution containing the magnetic microspheres is 4.8 × 10⁻⁶. 4 From 1.2 × 10⁻⁶ cells / μL to 1.2 × 10⁻ 5 The number of magnetic microspheres in the solution containing magnetic microspheres is 1-3 times the number of micropores.

2. The microsphere loading method according to claim 1, characterized in that, The microfluidic chip device also includes a glass slide disposed below the first layer, and the glass slide is bonded to the first layer.

3. The microsphere loading method according to claim 1, characterized in that, The microfluidic chip device also includes a magnet support, which is disposed below the chip and is detachably connected to the chip.

4. The microsphere loading method according to claim 1, characterized in that, The diameter of the inlet and the outlet is 1-3 mm, the length of the flow channel on either side of the detection area is 4-6 mm, the width and height of the flow channel are both 100-300 micrometers, the diameter of the detection area is 4-6 mm, the height of the detection area is 100-300 micrometers, and the diameter of the micropore array is 3-5 mm. The diameter of the micropore is 4-6 micrometers, the depth of the micropore is 3-5 micrometers, and the spacing between adjacent micropores is 2-4 micrometers.

5. The microsphere loading method according to claim 2, wherein the fabrication method of the microfluidic chip device comprises the following steps: S1: Prepare the second layer; S2: Prepare the first layer; and S3: The second layer is bonded to the first layer using oxygen plasma bonding, and the detection area of ​​the second layer is aligned with the micropore array of the first layer to obtain the chip.

6. The microsphere loading method according to claim 5, characterized in that, Step S1 includes the following steps: S1-1: Under the protection of a photomask, SU8-3050 photoresist is used to etch the mold onto a single-crystal silicon wafer, and wet photolithography is used to prepare the mold for casting. S1-2: Mix dimethylsiloxane and initiator at a mass ratio of (9-11):1, remove air bubbles by vacuuming, and pour into the mold obtained in step S1-1; and S1-3: After removing invisible air bubbles by vacuuming again, heat and cure. After removing the cured polydimethylsiloxane, punch holes at the inlet and outlet to obtain the second layer.

7. The microsphere loading method according to claim 5, characterized in that, Step S2 includes the following steps: S2-1: Under the protection of a photomask, SU8-3050 photoresist is used to etch the mold onto a single-crystal silicon wafer, and wet photolithography is used to prepare the mold for casting. S2-2: Mix dimethylsiloxane and initiator at a mass ratio of (9-11):1, remove air bubbles by vacuuming, and pour into the mold obtained in step S2-1; and S2-3: After removing invisible air bubbles by vacuuming again, heat to cure, and take out the cured polydimethylsiloxane to obtain the first layer.

8. The microsphere loading method according to claim 5, characterized in that, It also includes the following steps: S4: The chip is bonded to the glass slide using the oxygen plasma bonding method.

9. The microsphere loading method according to claim 1, characterized in that, The microsphere loading method further includes step 4: placing the chip loaded with the magnetic microspheres obtained in step 3 on a magnet support, so that the magnetic microspheres are magnetically connected to the magnet support, and the microfluidic chip device can be used for subsequent analysis.

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