Preparation and application of multi-volume sliding micro-fluidic chip

Abstract

The invention discloses preparation and application of a multi-volume sliding type micro-fluidic chip, and belongs to the technical field of micro-fluidic chips. The multi-volume sliding type micro-fluidic chip adopts an upper and lower double-layer stacked structure, the upper-layer chip is integrated with four fluid chambers with different volumes, the volume chambers are communicated with one another through a micro-channel network, and the tail end of each volume chamber is provided with a distribution outlet aligned with the lower-layer chip; array type micropore reaction areas are distributed on the surface of the lower-layer chip, and no internal channel exists. By rotating the upper-layer chip, the upper-layer chambers with different volumes can be accurately aligned with the lower-layer specific micropores, so that directional distribution of multi-volume liquid and independent control of the reaction area are realized. The micro-fluidic chip is simple in structure and easy and convenient to operate, the production and manufacturing cost is conveniently reduced, the complexity of fluid controlled release is reduced, the chip is compatible with biochemical detection and POCT scenes, and the chip is particularly suitable for PCR amplification experiments needing parallel processing of samples with multiple concentration gradients.

Description

Technical Field

[0001] This invention belongs to the field of microfluidic chip technology, and specifically relates to the fabrication and application of a multi-volume sliding microfluidic chip. Background Technology

[0002] A microfluidic chip is a chip that integrates basic functional units such as micrometer-scale channels and reaction cells on an operating platform. It is used in fields such as nucleic acid detection and cell culture in biology, chemistry, and medicine. Through precisely fabricated microchannels, micropumps, microvalves, and other structures on the chip, it is possible to control the flow of small amounts of liquid at a tiny scale.

[0003] Currently, the main technologies for nucleic acid diagnostics include nucleic acid hybridization, polymerase chain reaction (PCR), and isothermal amplification. Digital PCR (dPCR) is a third-generation PCR technology that can directly obtain the copy number of the target molecule, eliminating the need for standard curves or standards to determine the target copy number, thus enabling absolute quantification. This method disperses nucleic acid molecules into microwells through multiple dilutions and liquid separation until the number of molecules to be detected in each microwell does not exceed 1 (0 or 1). All samples in all microwells are amplified under the same conditions, producing a very strong fluorescence signal. The initial concentration of the target gene can be directly obtained by counting the positive droplets in the chambers.

[0004] The development of microfluidic technology has facilitated the maturity of digital PCR technology. However, this technology still faces limitations in nucleic acid detection: (I) The limited functionality and volume regulation of traditional microfluidic chips: Most existing microfluidic chips use a single-layer fixed structure, making it difficult to achieve independent storage and dynamic distribution of multi-volume reagents; (II) Limitations in chip fabrication materials: To ensure stable amplification of microdroplets, commonly used PDMS materials may cause moisture loss during amplification heating, necessitating the use of highly stable materials; (III) Traditional chips rely on external pump and valve systems for operation, resulting in bulky equipment that is difficult to meet the needs of portable detection.

[0005] The advantages of combining microfluidic chips with digital PCR are mainly as follows: (1) Absolute quantification: no standard curve needs to be established, and the results can be read directly; (2) Precise quantification: quantification can be performed down to a single copy; (3) Resistance to inhibition: by dividing the reaction solution into thousands of reaction units, the influence of inhibitors on amplification is effectively reduced. Studies have shown that dPCR has more precise quantification than qPCR. This technology has been applied in a wide range of fields such as gene mutation analysis, prenatal diagnosis of chromosomal abnormalities, copy number variation, pathogen detection, and transgenic detection. Summary of the Invention

[0006] To address the limitations of existing microfluidic chips, such as limited functionality, restricted volume adjustment, bulky devices that hinder portable testing, and the risk of contamination during opening, this invention aims to provide the preparation and application of a multi-volume sliding microfluidic chip for sensitive nucleic acid detection. Compared to existing technologies, this invention allows for direct microscopic counting and analysis of the microfluidic chip after isothermal incubation. Furthermore, the method of this invention is not only low-cost and simple to operate, but also enables accurate quantitative nucleic acid analysis.

[0007] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a multi-volume sliding microfluidic chip, mainly comprising an upper sliding microfluidic chip and a lower sliding microfluidic chip. Both the upper and lower sliding microfluidic chips are disk-shaped with the same diameter, and are tightly fitted together and can rotate around a central axis. A hydrophobic coating is provided on the contact surface of the upper and lower sliding microfluidic chips. Two to ten sets of microfluidic channels with the same structure are evenly distributed on the upper sliding microfluidic chip. Each set of microfluidic channels includes an inlet, an outlet, a liquid inlet microchannel, four different volumes of micropore assemblies, and a liquid outlet microchannel. The inlet is located at the edge of the upper sliding microfluidic chip, and the outlet is located at the upper sliding microfluidic chip. At the axis, between the inlet and outlet are sequentially connected liquid inlet microchannels, four micropore assemblies of different volumes, and liquid outlet microchannels. The micropore assemblies are arranged along the radial direction of the upper sliding microfluidic chip. The four micropore assemblies of different volumes are composed of four micropores of decreasing volume connected in series through a "pearl chain" channel. The lower sliding microfluidic chip is equipped with four micropore chamber groups of different volumes, arranged along the radial direction of the lower sliding microfluidic chip. The four micropore chamber groups of different volumes are composed of four micropores of decreasing volume. The micropores of the four micropore assemblies of different volumes on the upper sliding microfluidic chip and the micropores of the four micropore chamber groups of different volumes on the lower sliding microfluidic chip can overlap coaxially in the vertical direction.

[0008] Based on the above technical solution, the diameter of both the upper sliding microfluidic chip and the lower sliding microfluidic chip is 10~200 mm and the thickness is 1~3 mm.

[0009] Based on the above technical solution, the materials of the upper sliding microfluidic chip and the lower sliding microfluidic chip are one of PDMS, glass and PMMA.

[0010] Based on the above technical solution, the liquid inlet microchannel further branches into two groups of microchannels, which are then connected to two groups of four different volumes of micropore assemblies, and finally converge at the sample outlet through the liquid outlet microchannel.

[0011] Based on the above technical solution, further, the four microporous assemblies of different volumes are composed of micropores of 100~200 nL, 15~90 nL, 3~10 nL, and 0.5~2.5 nL connected in series through a "pearl chain" channel, with two micropores in each volume, and micropores of the same volume connected in series through the "pearl chain" channel; the four microporous chamber groups of different volumes are composed of micropores of 100~200 nL, 15~90 nL, 3~10 nL, and 0.5~2.5 nL, with no channels between the micropores, and two micropores in each volume.

[0012] Based on the above technical solution, the width of the "pearl chain" channel is 30~80 μm and the depth is 10~25 μm.

[0013] Secondly, the present invention provides a method for fabricating the above-mentioned multi-volume sliding microfluidic chip, comprising the following steps: (1) Fabrication of the upper sliding microfluidic chip 1: 1) Preparation of the primary etching substrate: A smooth, scratch-free borosilicate glass plate is selected as the substrate; a chromium layer with a thickness of approximately 100-150 nm is deposited on the clean and dry glass substrate surface by magnetron sputtering as a hard mask; then a layer of positive photoresist is spin-coated onto the chromium layer; the pre-prepared first photomask is precisely aligned with the photoresist-coated substrate. The first photomask contains a designed micro-hole array pattern (including 100-200 nL micro-holes, 15-90 nL micro-holes, 3-10 nL micro-holes, etc.). (Four volumetric pore structures: nL micropores and 0.5~2.5nL micropores); contact exposure is performed using a UV exposure machine; subsequently, the substrate is immersed in the corresponding photoresist developer to dissolve and remove the photoresist in the exposed areas; the developed substrate is then immersed in a chromium etching solution until the chromium layer exposed in the etching solution is completely removed, exposing the underlying glass surface, while the chromium layer in the photoresist-covered areas remains intact; after etching, the substrate is immediately rinsed with deionized water to terminate the reaction and dried with nitrogen; the etched substrate is then immersed in a photoresist stripping solution to thoroughly remove the residual photoresist layer, rinsed again with deionized water and ethanol, and dried with nitrogen; a micropore array pattern mask is formed on the glass substrate; 2) Secondary Etching (Deep Etching of Glass Substrate): On the substrate obtained in step 1), a second photomask is precisely aligned with the chromium mask pattern on the substrate for ultraviolet exposure. This second photomask defines the microfluidic channels and connection paths, except for the micro-holes. The substrate is then developed in the corresponding developer to remove the photoresist in the exposed area of ​​the second photomask, revealing the chromium layer in the area to be etched. The substrate is rinsed with deionized water and dried with nitrogen. It is then immersed in a glass etching solution with a target etching depth of 10-25 micrometers. Once the target depth is reached, the substrate is immediately removed from the etching solution to terminate the etching reaction. Subsequently, the substrate is ultrasonically cleaned 1-10 times with deionized water and ethanol, and then immersed in a chromium etching solution to remove the remaining chromium mask layer. Finally, the substrate is thoroughly cleaned with deionized water and ethanol and dried with nitrogen. At this point, microstructures with different depths (the depth at the micro-holes is determined by the primary etching, while the depth at the channels, etc., is 10-25 micrometers) are formed on the glass substrate. 3) Surface functionalization treatment: The glass substrate etched in step 2) is placed in the following solvents for ultrasonic cleaning in sequence: chloroform: ultrasonic for 10 minutes to remove organic contaminants; acetone: ultrasonic for 10 minutes; deionized water: ultrasonic for 10 minutes; anhydrous ethanol: ultrasonic for 10 minutes; then the cleaned and dried substrate is placed in a plasma cleaner for oxygen plasma treatment. Finally, the plasma-treated substrate is transferred to a vapor deposition chamber for vapor phase silanization treatment. After the treatment is completed, the substrate is taken out and baked at 100~150°C for 20~60 minutes. (2) Fabrication of the lower sliding microfluidic chip: The fabrication of the lower sliding microfluidic chip is the same as the fabrication of the upper sliding microfluidic chip in step 1), except that in step 2), a lower chip photomask without channels and sample inlet / outlet holes is used for deep etching in the secondary etching. (3) Assembly of multi-volume sliding microfluidic chip: Immerse the lower sliding microfluidic chip in the degassed oil phase, align the upper sliding microfluidic chip with the patterned surface and place it on the lower sliding microfluidic chip; after accurately aligning the positions of the two plates, fix them with a fixing clip, and drill the sample inlet and the central sample outlet around the upper sliding microfluidic chip to obtain the chip.

[0014] Based on the above technical solution, further, the developing solution mentioned in step 1) is prepared by mixing AZ 400K and water in a ratio of 1:4; the chromium etching solution is a mixed aqueous solution of 0.6 mol / L perchloric acid and 0.365 mol / L cerium ammonium nitrate; the soaking temperature is 70~80℃, and the soaking time is 10~15 minutes.

[0015] Based on the above technical solution, further, the glass etching solution mentioned in step 2) is a mixed aqueous solution composed of hydrofluoric acid, ammonium fluoride and nitric acid in a molar ratio of 1:0.5:0.75; the etching temperature is controlled at 30-40℃; the chromium etching solution is a mixed aqueous solution of 0.6mol / L perchloric acid and 0.365mol / L cerium ammonium nitrate.

[0016] Based on the above technical solution, further, in step 3), after each ultrasonic treatment, the substrate is rinsed with fresh corresponding solvent; finally, the substrate surface is dried with high-purity nitrogen gas. The specific process of oxygen plasma treatment is as follows: a clean and dry substrate is placed in the reaction chamber of a plasma cleaner, high-purity oxygen is introduced, the chamber pressure is maintained at 0.2~0.5 mbar, and oxygen plasma treatment is performed for 120 seconds. The specific process of vapor phase silanization is as follows: dimethyl dichlorosilane ((CH3)2SiCl2, DMDCS) vapor reacts with the substrate surface at room temperature for 0.5 to 2 hours.

[0017] Based on the above technical solution, further, the oil phase in step (3) is one or more of fluorinated oil, mineral oil, silicone oil and tetradecane.

[0018] Thirdly, the present invention provides the application of the above-mentioned multi-volume sliding microfluidic chip in nucleic acid detection.

[0019] Based on the above technical solution, the nucleic acid detection method further includes PCR amplification.

[0020] Compared with the prior art, the present invention has the following beneficial effects: The multi-volume sliding microfluidic chip of this invention uses a rotary volume switching (10° increments) to replace the traditional sliding structure, reducing frictional loss and extending service life; the dual-layer functional decoupling design focuses on multi-reagent dispensing in the upper layer and on reaction detection in the lower layer, reducing the risk of cross-contamination; at the same time, the droplet microfluidic chip integrates generation and detection, and there is no opening step in the detection process, eliminating the risk of cross-contamination. These measures will improve the success rate and timeliness of digital PCR implementation. Attached Figure Description

[0021] To more clearly illustrate the embodiments of the present invention, the accompanying drawings involved in the embodiments will be briefly described below.

[0022] Figure 1This is a schematic diagram of the structure of the multi-volume sliding microfluidic chip in Example 1, wherein 1: upper sliding microfluidic chip, 2: lower sliding microfluidic chip, 3: sample inlet, 4: sample outlet, 5: 125 nL micropore, 6: 25 nL micropore, 7: 5 nL micropore, 8: 1 nL micropore, 9: liquid inlet microchannel, 10: liquid outlet microchannel.

[0023] Figure 2 This is a top view of the multi-volume sliding microfluidic chip after combining the upper sliding microfluidic chip and the lower sliding microfluidic chip in Example 1.

[0024] Figure 3 This is a flowchart of the liquid transfer process verified by a multi-volume sliding microfluidic chip using different colored dyes in Example 2.

[0025] Figure 4 This is a flowchart of nucleic acid detection based on a multi-volume sliding microfluidic chip in Example 3.

[0026] Figure 5 This is a graph showing the fluorescence results of detecting ovarian cancer miRNA (A) and circRNA (B) based on a multi-volume sliding microfluidic chip in Example 4.

[0027] Figure 6 This is a graph showing the fitting curve results of ovarian cancer circRNA (A) and miRNA (B) detected based on a multi-volume sliding microfluidic chip in Example 4.

[0028] Figure 7 This is a graph showing the results of detecting the proportion of micropores of different volumes based on a multi-volume sliding microfluidic chip in Example 4. Detailed Implementation

[0029] The present invention will be described in detail below with reference to the embodiments. However, the implementation of the present invention is not limited thereto. Obviously, the embodiments described below are only some embodiments of the present invention. For those skilled in the art, other similar embodiments can be obtained without creative effort and all fall within the protection scope of the present invention.

[0030] Unless otherwise specified, the methods used in the following technical solutions are conventional methods, and the materials and reagents used are commercially available unless otherwise specified.

[0031] Example 1: Fabrication of a multi-volume sliding microfluidic chip This embodiment provides a multi-volume sliding microfluidic chip, see [link]. Figure 1-2The system mainly consists of an upper sliding microfluidic chip 1 and a lower sliding microfluidic chip 2. Both the upper and lower sliding microfluidic chips 1 and 2 are disc-shaped with the same dimensions: a diameter of 90 mm and a thickness of 2 mm. The upper and lower sliding microfluidic chips 1 and 2 are tightly fitted together and can rotate around a central axis. A nanoscale hydrophobic coating is provided on the contact surface of the upper and lower sliding microfluidic chips 1 and 2. The upper sliding microfluidic chip 1 is made of transparent soda-lime glass. Four sets of microfluidic channels with the same structure are evenly distributed on the upper sliding microfluidic chip 1. Each set of microfluidic channels includes an inlet 3, an outlet 4, a liquid inlet microchannel 9, four different volumes of micropore combinations, and an outlet microchannel 10. Located at the edge of the upper sliding microfluidic chip 1, the sample outlet 4 is located on the axis of the upper sliding microfluidic chip 1. Between the sample inlet 3 and the sample outlet 4 are sequentially connected liquid inlet microchannel 9, four micropore assemblies of different volumes, and liquid outlet microchannel 10. The micropore assemblies are arranged along the radial direction of the upper sliding microfluidic chip 1. The liquid inlet microchannel 9 branches into two groups of microchannels, which are then connected to two groups of four micropore assemblies of different volumes, and finally converge at the sample outlet 4 via the liquid outlet microchannel 10. The four micropore assemblies of different volumes are connected by a 125 Micropores 5, 25 nL, 6, 7, and 8 are connected in series via a "pearl chain" channel. Each volume contains two micropores connected in series via the "pearl chain" channel. The upper sliding microfluidic chip 1 has 32 micropores of each volume. The "pearl chain" channel is 50 μm wide and 20 μm deep. The lower sliding microfluidic chip 2 has 16 groups of four different volumes of micropore chambers evenly arranged along the radial direction of the lower sliding microfluidic chip 2. The four different volumes of micropore chamber groups consist of 125 nL, 25 nL, 5 nL, and 8 micropores. The microfluidic chip consists of nL micropores, with two micropores per volume. There are no channels between the micropores. The micropores of the four different volumes of the upper sliding microfluidic chip 1 and the micropores of the four different volumes of the lower sliding microfluidic chip 2 can overlap coaxially in the vertical direction. The lower sliding microfluidic chip 2 also has 32 micropores of each volume.

[0032] The inlet 3 is used to provide power support for the solution inside the chip. One end of it is connected to the chip channel and the other end is connected to the syringe. The closed micropores generated by the multi-volume sliding microfluidic chip, which is composed of the upper sliding microfluidic chip 1 and the lower sliding microfluidic chip 2, serve as micropores that integrate liquid mixing, amplification and detection.

[0033] The multi-volume sliding microfluidic chip disclosed in this invention integrates a droplet generation and storage module, a nucleic acid amplification module, and a detection module into one unit. It is an applicable platform that can detect a variety of nucleic acid biomarkers.

[0034] The fabrication method of the above-mentioned multi-volume sliding microfluidic chip, wherein the upper sliding microfluidic chip 1 includes the following steps: (1) Preparation of primary etching substrate: A borosilicate glass plate with a smooth surface and no scratches is selected as the substrate. A chromium (Cr) layer with a thickness of about 100~150 nm is deposited on the clean and dry glass substrate surface by magnetron sputtering as a hard mask. Subsequently, a layer of positive photoresist is spin-coated on the chromium layer. The pre-prepared first photomask is precisely aligned with the photoresist-coated substrate. The photomask contains a designed micro-hole array pattern (including four volumetric hole structures: 1 nL, 5 nL, 25 nL, and 125 nL). Contact exposure is performed using an ultraviolet exposure machine. Subsequently, the substrate is immersed in the corresponding photoresist developer (AZ400K diluted with water at a ratio of 1:4) to dissolve and remove the photoresist in the exposed areas. The developed substrate is immersed in a chromium etching solution, which is a mixed aqueous solution of 0.6 mol / L perchloric acid (HClO4) and 0.365 mol / L cerium ammonium nitrate ((NH4)2Ce(NO3)6). Etching is performed at room temperature until the chromium layer exposed in the etching solution is completely removed, exposing the underlying glass surface, while the chromium layer in the photoresist-covered area remains intact. The etching time is typically 1-3 minutes. After etching, the substrate is immediately rinsed with plenty of deionized water to terminate the reaction and dried with nitrogen. The etched substrate is then immersed in a photoresist stripping solution and heated (70-80°C) for 10-15 minutes to thoroughly remove the residual photoresist layer. It is then rinsed again with deionized water and ethanol and dried with nitrogen. At this point, a micro-hole array pattern mask is formed on the glass substrate.

[0035] (2) Secondary etching (deep etching of glass substrate): On the substrate after step (1), a second photomask is precisely aligned with the chromium mask pattern on the substrate for ultraviolet exposure. The second photomask defines the microfluidic channels and connection paths, except for the micropores. The substrate is developed in the corresponding developer to remove the photoresist in the exposed area of ​​the second photomask and expose the chromium layer in the area to be etched. The substrate is rinsed with deionized water and dried with nitrogen. The substrate is then immersed in a glass etching solution. The etching solution is a mixed aqueous solution of hydrofluoric acid (HF), ammonium fluoride (NH4F), and nitric acid (HNO3) in a molar ratio of 1:0.5:0.75. The etching is carried out in a constant temperature water bath at a temperature of 30-40°C to stabilize the etching rate. The target etching depth is 20 micrometers. After reaching the target depth, the substrate is immediately removed from the etching solution and immersed in a large amount of flowing deionized water for at least 5 minutes to rapidly dilute and terminate the etching reaction. Subsequently, the substrate is ultrasonically cleaned several times with deionized water and ethanol to thoroughly remove residual etching solution and reaction products. The substrate is immersed in chromium etching solution (same as step 1) to remove the remaining chromium mask layer; then, the substrate is thoroughly cleaned with deionized water and ethanol and dried with nitrogen; at this time, microstructures with different depths (the depth at the micropores is determined by the primary etching, and the depth at the channels, etc. is 20 micrometers) are formed on the glass substrate. (3) Surface functionalization treatment: The etched glass substrate is placed in the following solvents for ultrasonic cleaning in sequence: Chloroform: ultrasonic for 10 minutes to remove organic contaminants; Acetone: ultrasonic for 10 minutes to remove grease and residual photoresist; Deionized water (DI Water): ultrasonic for 10 minutes to remove water-soluble impurities; Anhydrous ethanol (Ethanol): ultrasonic for 10 minutes to dehydrate and replace; After each ultrasonic treatment, the substrate is rinsed with fresh corresponding solvent; Finally, the substrate surface is carefully dried with high-purity nitrogen (N2); Oxygen plasma treatment (hydrophilization): The clean and dry substrate is placed in the reaction chamber of the plasma cleaner; High-purity oxygen (O2) is introduced, and the chamber pressure is maintained at 0.2-0.5 mbar; Radio frequency power is applied for oxygen plasma treatment for 120 seconds; This process effectively removes residual organic contaminants and generates a large number of hydroxyl groups (-OH) on the glass surface, significantly enhancing its hydrophilicity; Vapor phase silanization (hydrophobicization): The plasma-treated substrate is rapidly transferred to a vacuum dryer or a dedicated vapor deposition chamber, where dimethyl dichlorosilane ((CH3)2SiCl2, DMDCS) vapor reacts with the substrate surface at room temperature (~25°C) for 1 hour. The chlorosilane groups (-SiCl2) in the DMDCS molecules undergo a condensation reaction with the hydroxyl groups (-OH) on the glass surface, forming strong Si-O-Si covalent bonds and leaving hydrophobic methyl (-CH3) groups. Post-treatment: After the reaction, an inert gas (such as nitrogen or argon) is slowly introduced into the chamber until atmospheric pressure is reached. The substrate is then removed and placed in an oven at 120°C for 30 minutes to promote cross-linking between silane molecules and remove physically adsorbed silane monomers. After cooling to room temperature, the substrate surface should exhibit stable hydrophobicity. (4) Chip Regeneration: Removal of Organic Residues (Piranha Cleaning): When the chip needs to be regenerated after use due to contamination by organic pollutants (such as proteins and oils), immerse the chip in a freshly prepared Piranha solution. The Piranha solution is a mixture of concentrated sulfuric acid (H2SO4, 95-98%) and hydrogen peroxide (H2O2, 30%) in a 3:1 volume ratio; soak at room temperature for 15-30 minutes; the Piranha solution can efficiently oxidize and remove almost all organic residues; Thorough Cleaning: Remove the chip from the Piranha solution and rinse it with plenty of deionized water; then ultrasonically clean it with deionized water and ethanol for at least 10 minutes each to ensure complete removal of all acid and oxidation products; finally, dry it with high-purity nitrogen; Surface Functionalization Restoration: Repeat step (3) completely with the thoroughly cleaned and dried chip. The process includes "oxygen plasma treatment (120 seconds)" and "vapor phase dimethyl dichlorosilane deposition (1 hour)" as well as the subsequent baking process. This process removes trace contaminants remaining after regeneration and cleaning, reactivates surface hydroxyl groups, and redeposits a hydrophobic silane layer, restoring the hydrophilic / hydrophobic properties of the chip surface to near their initial state, thereby enabling chip regeneration and reuse. The lower sliding microfluidic chip 2 has the same fabrication process as the upper sliding microfluidic chip 1, except that it does not include the secondary etching step. Fabrication of the lower sliding microfluidic chip: The fabrication of the lower sliding microfluidic chip is the same as step 1) of the fabrication of the upper sliding microfluidic chip, except that in step 2), a lower chip photomask without channels and sample inlet / outlet holes is used for deep etching in the secondary etching. (5) The assembly and operation of the multi-volume sliding microfluidic chip are all completed in a degassed oil phase environment (volume ratio of mineral oil to tetradecane = 1:4); the key operation steps include: after immersing the lower sliding microfluidic chip into the oil phase, aligning and placing the upper sliding microfluidic chip with the patterned surface on the lower sliding microfluidic chip; accurately aligning the double plate positions and fixing them with a snap-fit ​​clip; drilling four sample inlets and one central sample outlet around the upper sliding microfluidic chip 1 to obtain the chip.

[0036] Example 2: Verification of liquid transfer using a multi-volume sliding microfluidic chip with different colored dyes This embodiment uses different colored dyes to verify bright-field microscopic images of liquid transfer via a multi-volume sliding microfluidic chip, such as... Figure 3 As shown, the operation process is as follows: (i) injection of orange dye for microchannel visualization; (ii) first-stage droplet transfer; (iii) loading of blue dye; (iv) controlled droplet fusion (yellow / blue = 1:1 volume ratio), demonstrating the successful implementation of multi-volume microfluidic chip rotational transfer of liquid.

[0037] Example 3: A method for detecting nucleic acids based on a multi-volume sliding microfluidic chip This embodiment integrates the multi-volume sliding microfluidic chip prepared in Example 1 with the established isothermal amplification CRISPR technology (Chinese Invention Patent Application No.: 2025102123288) for nucleic acid detection. Figure 4 As shown, the specific steps include: Step 1: Before use, the micropores of the upper sliding microfluidic chip 1 and the lower sliding microfluidic chip 2 are staggered (10°) and do not overlap. The reagent solution sample (rolling circle amplification related reagent) is injected into each injection port by pipetting. The channel is loaded synchronously by applying a standard pressure of 18 mmHg. The rolling circle amplification (RCA) reaction reagent is injected into each micropore of the micropore assembly of the upper sliding microfluidic chip 1 through the continuous "pearl chain" microfluidic channel to form microdroplets. Step 2: By rotating the upper sliding microfluidic chip 1 by 10°, the sample from the upper sliding microfluidic chip 1 flows into the target microwell 2 of the lower sliding microfluidic chip 2. After a simple sliding operation to align the channels of the upper and lower chips, the droplets are transferred to the channels of the lower chip. The chip is then placed in a PCR instrument equipped with a heating module and incubated at 48°C for 15 minutes. Step 3: After resetting the upper sliding microfluidic chip 1, inject CRISPR / Cas12a cleavage system reagents into each microwell of the microwell assembly of the upper sliding microfluidic chip 1, and use a second sliding operation to fuse the CRISPR / Cas12a reagent droplets formed in each microwell of the upper sliding microfluidic chip 1 with the amplification products in the microwell of the lower sliding microfluidic chip 2, and incubate at 37°C for 15 minutes; Step 4: Fluorescence imaging was performed before and after incubation using a Nikon Ti2 microscope. Automatic image stitching and analysis were completed using NIS-Elements software (v5.01). The digital average fluorescence intensity of each channel was measured. Green fluorescence indicated a positive result, confirming it as the target sample. No color change in the remaining reaction cells indicated a negative result. Figure 4 Finally, the copy number is calculated using the most probable number (MPN) likelihood function theory.

[0038] A nanoscale hydrophobic coating is provided between the upper sliding microfluidic chip 1 and the lower sliding microfluidic chip 2, forming a hydrophilic pathway only when aligned with the micropores to prevent reagent diffusion and contamination. A multi-chamber sliding microfluidic chip with gradient volume adaptive function was developed. This chip system uses a modularly designed 4-level microchamber array (1nL-125 nL) to achieve dynamic sample volume adaptation. This multi-volume chip consists of two layers, each containing four different chamber volumes of 125 nL, 25 nL, 5 nL, and 1 nL. The large-volume chambers (125 nL and 25 nL) are used to capture low-abundance targets, while the small-volume chambers (5 nL and 1 nL) are used to analyze high-concentration samples. Each volume specification corresponds to 64 chambers.

[0039] Example 4: A method for detecting circRNA and miRNA biomarkers in ovarian cancer based on a multi-volume sliding microfluidic chip. Referring to the usage steps of Example 3, five orders of magnitude dilution gradients (from 10) were used. -1 10 0 10 1 10 2 10 3 The detection of synthetic circRNA (hsa_circ_0049101) and miRNA (hsa-miR-338-3p) templates within the range of copies / µL (Chinese Invention Patent Application No.: 2025102123288) specifically includes the following steps: Step 1: Template Synthesis: The synthesis steps for circular RNA (circRNA) are as follows: First, linear RNA is transcribed in vitro from a double-stranded DNA (dsDNA) template using T7 RNA polymerase; then, a circularization reaction is performed using T4 RNA ligase 2 and DNA clip primers; the linear RNA (1 µM) and clip primers (5 µM) are annealed at 65°C for 5 minutes, followed by a gradient cooling to 12°C at a rate of 1°C / min; T4 RNA ligase 2 and an RNase inhibitor are added for ligation, incubated at 37°C for 90 minutes, and then inactivated by heating at 80°C for 5 minutes; to remove residual linear RNA, the product is treated with RNase R at 37°C for 30 minutes and then inactivated at 70°C for 10 minutes; excess DNA clip primers are digested with RNase-free DNase I at 37°C for 30 minutes. Finally, purification is performed using the Monarch® RNA purification kit according to the manufacturer's instructions; the miRNA template is directly synthesized by Nanjing GenScript Biotech Co., Ltd.

[0040] Step 2: Sample and Amplification Reagent Loading: All RNA samples were thawed on ice before use, biological reagents were thawed on ice, and chemical solutions were equilibrated to room temperature. The detection procedure included: 1 μL serially diluted circRNA / miRNA template, 1 μL RNase inhibitor, 2 μL pre-circular amplification primers (100 nM), and 2.4 μL magnesium sulfate (100 mM); Circular amplification: 2 μL circular DNA scaffold (40 nM), 1 μL dNTP mixture (2.5 mM each), 1 μL φ29 DNA polymerase (2 U / μL), 2 μL 10× reaction buffer, and nuclease-free water to a final volume of 20. A final volume of μL was injected into the injection port of the above mixture. The liquid was then injected into the rolling circle amplification (RCA) reaction reagent through a continuous "bead chain" microfluidic channel using negative pressure. Microdroplets were formed in the channels of the upper chip. After aligning the channels of the upper and lower chips with a simple sliding operation, the microdroplets were transferred to the channels of the lower chip. The chip was then placed in a PCR instrument equipped with a custom heating module and incubated at 48°C for 15 minutes.

[0041] Step 3: CRISPR / Cas12a Reagent Loading: After the upper chip is reset, inject the CRISPR / Cas12a cleavage system reagent; then, introduce the CRISPR / Cas12a reagent into the channels of the upper chip to form microdroplets, and use a two-stage sliding operation to align and fuse them with the amplification products in the channels of the lower chip. Incubate at 37°C for 15 minutes. Fluorescence imaging was performed before and after incubation using a Nikon Ti2 microscope, and automatic image stitching and analysis were achieved using NIS-Elements software (v5.01). Copy number calculation was based on the most probable number (MPN) likelihood function theory. The endpoint fluorescence value was detected using an Infinite® 200 PRO microplate reader, and fluorescence kinetics were monitored using the QuantStudio™ 3 real-time PCR system.

[0042] This study improves the computational method for microfluidic chips. Specifically, the RNA concentration determination method is based on the most probable number (MPN) likelihood function theory. The concentration λ (copies / µL) is determined by substituting the experimental results of each test volume (i=1,2,3,...) into Equation 1, where ni, ki, and vi represent the total number of reaction chambers, the number of positive reaction chambers, and the reaction chamber volume (µL) for each volume i, respectively. The relative contribution of each reaction chamber volume is determined by calculating the total uncertainty σ using Equation 2, which is based on Fisher's information theory. (1) (2) Figure 5Figures A and 5B show the endpoint fluorescence images for circRNA and miRNA detection using a multi-volume microfluidic chip method. The proportion of positive microcavities in each volume group was statistically analyzed, and the template concentration was calculated using formula (1). No false positives were observed after amplification in the negative control experiment. The number of fluorescently positive microcavities increased accordingly with increasing RNA template concentration.

[0043] The fitted curve is shown below. Figure 6 As shown, the measured concentration gradient of circRNA was 1.296 × 10⁻⁶. 3 1.124×10 2 1.98×10 1 4.02×10 0 The concentration of 0.455 copies / μL was used for miRNA, while the concentration was 1.356 × 10⁻⁶ copies / μL. 3 2.357×10 2 1.23×10 1 1.23×10 0 The detection limits for circRNA and miRNA were 0.128 copies / μL; the detection limits for circRNA and miRNA were 1.23 and 4.02 copies / μL, respectively; hsa_circ_0049101 (logY = 0.804logX + 0.694 (R 2 = 0.995)) and hsa-miR-338-3p (logY = 0.882logX + 0.475 (R 2 The input concentration (= 0.997) showed a significant linear correlation with the calculated concentration. Notably, all gradient experimental data were in high agreement with the theoretical distribution of the predicted concentration of the input RNA. Within the dynamic range detected by this multi-volume microfluidic chip, the contribution of different microcavities to RNA concentration calculation varies. The results are as follows... Figure 7 The results show that as the concentration of the control RNA template increases, the microcavity that dominates the final concentration calculation gradually transitions from a large volume (125 nL) to a medium volume (25 nL and 5 nL), and finally to a small volume microcavity (1 nL). The relative contribution of each microcavity volume is quantified by its percentage weight in the total uncertainty (σ) of Equation (2). These results demonstrate that this multi-volume microfluidic chip performs exceptionally well in detecting low-concentration RNA. Through multi-volume design, this system significantly expands the dynamic range of RNA detection while maintaining high specificity and excellent anti-interference capabilities.

[0044] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; 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 or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A multi-volume sliding microfluidic chip, characterized in that, The multi-volume sliding microfluidic chip mainly comprises an upper sliding microfluidic chip and a lower sliding microfluidic chip. Both the upper and lower sliding microfluidic chips are disk-shaped with the same diameter, and are tightly fitted together, allowing them to rotate around a central axis. A hydrophobic coating is provided on the contact surface of the upper and lower sliding microfluidic chips. Two to ten sets of identical microfluidic channels are evenly distributed on the upper sliding microfluidic chip. Each set of microfluidic channels includes an inlet, an outlet, a liquid inlet microchannel, four different volumes of micropore assemblies, and an outlet microchannel. The inlet is located at the edge of the upper sliding microfluidic chip, and the outlet is located at the axis of the upper sliding microfluidic chip. Between the inlet and outlet are sequentially connected liquid inlet microchannels, four micropore assemblies of different volumes, and liquid outlet microchannels. The micropore assemblies are arranged along the radial direction of the upper sliding microfluidic chip. The four micropore assemblies of different volumes are composed of four micropores of decreasing volume connected in series through a "pearl chain" channel. The lower sliding microfluidic chip has four micropore chamber groups of different volumes arranged along the radial direction of the lower sliding microfluidic chip. The four micropore chamber groups of different volumes are composed of four micropores of decreasing volume. The micropores of the four micropore assemblies of different volumes on the upper sliding microfluidic chip and the micropores of the four micropore chamber groups of different volumes on the lower sliding microfluidic chip can overlap coaxially in the vertical direction.

2. The multi-volume sliding microfluidic chip according to claim 1, characterized in that, The diameter of both the upper sliding microfluidic chip and the lower sliding microfluidic chip is 10~200 mm, and the thickness is 1~3 mm. The materials of the upper sliding microfluidic chip and the lower sliding microfluidic chip are one of PDMS, glass and PMMA.

3. The multi-volume sliding microfluidic chip according to claim 1, characterized in that, The inlet microchannel branches into two sets of microchannels, which are then connected to two sets of four different types of micropore assemblies. Finally, they converge at the outlet via the outlet microchannel.

4. The multi-volume sliding microfluidic chip according to claim 1, characterized in that, The four types of microporous assemblies with different volumes are composed of micropores of 100~200 nL, 15~90 nL, 3~10 nL, and 0.5~2.5 nL connected in series via a "pearl chain" channel. Each volume contains two micropores, and micropores of the same volume are connected in series via the "pearl chain" channel. The four types of microporous chamber groups with different volumes are composed of micropores of 100~200 nL, 15~90 nL, 3~10 nL, and 0.5~2.5 nL. There are no channels between the micropores, and each volume contains two micropores.

5. The multi-volume sliding microfluidic chip according to claim 1, characterized in that, The width of the "pearl chain" channel is 30~80 μm and the depth is 10~25 μm.

6. The method for fabricating a multi-volume sliding microfluidic chip according to any one of claims 1-5, characterized in that, Includes the following steps: (1) Fabrication of the upper sliding microfluidic chip 1: 1) Preparation of primary etching substrate: Select a borosilicate glass plate with a smooth surface and no scratches as the substrate; A chromium layer with a thickness of about 100~150 nm is deposited as a hard mask on the surface of a clean and dry glass substrate by magnetron sputtering. Subsequently, a layer of positive photoresist is spin-coated onto the chromium layer; the pre-prepared first photomask is precisely aligned with the photoresist-coated substrate, the first photomask containing the designed micro-hole array pattern; Contact exposure was performed using a UV exposure machine; Subsequently, the substrate is immersed in the corresponding photoresist developer to dissolve and remove the photoresist in the exposed areas; The developed substrate is immersed in a chromium etching solution until the chromium layer exposed in the etching solution is completely removed, exposing the underlying glass surface, while the chromium layer in the photoresist-covered area remains intact. After etching is completed, immediately rinse the substrate with deionized water to terminate the reaction and dry it with nitrogen gas; The etched substrate was immersed in photoresist stripping solution to completely remove the residual photoresist layer. It was then rinsed again with deionized water and ethanol and dried with nitrogen. A micro-hole array pattern mask was formed on the glass substrate. 2) Secondary etching: On the substrate obtained in step 1), a second photomask is precisely aligned with the chromium mask pattern on the substrate for ultraviolet exposure. The second photomask defines the microfluidic channels and connection paths, except for the micropores. The substrate is then developed in the corresponding developer to remove the photoresist in the exposed area of ​​the second photomask, revealing the chromium layer in the area to be etched. The substrate is then rinsed with deionized water and dried with nitrogen. The substrate is immersed in the glass etching solution; the target etching depth is 10-25 micrometers; after the target depth is reached, the substrate is immediately removed from the etching solution to terminate the etching reaction. Subsequently, the substrate was ultrasonically cleaned 1 to 10 times with deionized water and ethanol, and then immersed in chromium etching solution to remove the remaining chromium mask layer. After that, the substrate was thoroughly cleaned with deionized water and ethanol and dried with nitrogen. At this point, microstructures of different depths were formed on the glass substrate. 3) Surface functionalization treatment: The glass substrate etched in step 2) is placed in the following solvents for ultrasonic cleaning in sequence: chloroform: ultrasonic for 10 minutes to remove organic contaminants; acetone: ultrasonic for 10 minutes; deionized water: ultrasonic for 10 minutes; anhydrous ethanol: ultrasonic for 10 minutes; then the cleaned and dried substrate is placed in a plasma cleaner for oxygen plasma treatment. Finally, the plasma-treated substrate is transferred to a vapor deposition chamber for vapor phase silanization treatment. After the treatment is completed, the substrate is taken out and baked at 100~150°C for 20~60 minutes. (2) Fabrication of the lower sliding microfluidic chip: The fabrication of the lower sliding microfluidic chip is the same as the fabrication of the upper sliding microfluidic chip in step 1), except that in step 2), a lower chip photomask without channels and sample inlet / outlet holes is used for deep etching in the secondary etching. (3) Assembly of multi-volume sliding microfluidic chip: Immerse the lower sliding microfluidic chip in the degassed oil phase, align the upper sliding microfluidic chip with the patterned surface and place it on the lower sliding microfluidic chip; after accurately aligning the positions of the two plates, fix them with a fixing clip, and drill the sample inlet and the central sample outlet around the upper sliding microfluidic chip to obtain the chip.

7. The preparation method according to claim 6, characterized in that, The developing solution mentioned in step 1) is prepared by mixing AZ 400K and water in a ratio of 1:4; the chromium etching solution is a mixed aqueous solution of 0.6 mol / L perchloric acid and 0.365 mol / L cerium ammonium nitrate; the soaking temperature is 70~80℃ and the soaking time is 10~15 minutes; The glass etching solution mentioned in step 2) is a mixed aqueous solution composed of hydrofluoric acid, ammonium fluoride and nitric acid in a molar ratio of 1:0.5:0.75; the etching temperature is controlled at 30-40℃; the chromium etching solution is a mixed aqueous solution of 0.6 mol / L perchloric acid and 0.365 mol / L cerium ammonium nitrate.

8. The preparation method according to claim 6, characterized in that, After each ultrasonic treatment in step 3), the substrate is rinsed with the corresponding fresh solvent; finally, the substrate surface is dried with high-purity nitrogen. The specific process of oxygen plasma treatment is as follows: a clean and dry substrate is placed in the reaction chamber of a plasma cleaner, high-purity oxygen is introduced, the chamber pressure is maintained at 0.2~0.5 mbar, and oxygen plasma treatment is performed for 120 seconds. The specific process of vapor phase silanization is as follows: dimethyl dichlorosilane ((CH3)2SiCl2, DMDCS) vapor reacts with the substrate surface at room temperature for 0.5 to 2 hours.

9. The preparation method according to claim 6, characterized in that, The oil phase mentioned in step (3) is one or a combination of two or more of fluorinated oil, mineral oil, silicone oil and tetradecane.

10. The application of the multi-volume sliding microfluidic chip according to any one of claims 1-5 in nucleic acid detection.

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