Constant-speed electrophoresis micro-fluidic chip

Abstract

The invention relates to the technical field of constant-speed electrophoresis, and discloses a constant-speed electrophoresis micro-fluidic chip which comprises a glass substrate and a PDMS flow channel bonded on the glass substrate, the constant-speed electrophoresis micro-fluidic chip sequentially comprises a TE electrode area, a main flow channel area and an LE electrode area in the fluid path direction, open holes are formed in the positions above the TE electrode area and the LE electrode area, and the main flow channel area is communicated with the TE electrode area. The plunger with the PDMS compact film is fixed in the TE electrode area and the LE electrode area through holes, an external electrode is inserted into the TE electrode area and the LE electrode area from the two sides of the PDMS flow channel, the position after insertion is right opposite to the PDMS compact film below the plunger, preset negative pressure is always kept after electrolyte and a sample are injected into the PDMS flow channel above the plunger, and then the external electrode is inserted into the TE electrode area and the LE electrode area; long and narrow pipeline arrays are arranged between the TE electrode area and the LE electrode area and the main flow channel area, all pipelines are arranged in parallel, the area of the cross section of each pipeline is smaller than that of the cross section of the main flow channel area, and the constant-speed electrophoresis stability is higher.

Description

Technical Field

[0001] This invention relates to the field of isokinetic electrophoresis technology, and in particular to an isokinetic electrophoresis microfluidic chip. Background Technology

[0002] With the rapid development of life sciences and clinical medicine, trace analysis of extremely low concentrations of components in biological samples has become a key tool for genomics research, early cancer screening, and diagnosis of infectious diseases. In the field of nucleic acid detection, the accurate extraction and quantification of nucleic acids from trace, rare, or highly degraded samples (such as FFPE formalin-fixed paraffin-embedded samples) is of paramount importance for improving the accuracy and reliability of subsequent gene sequencing. The mainstream techniques for nucleic acid sample preparation mainly rely on manual or semi-automated centrifugation, column extraction, and magnetic bead methods. While these techniques can accomplish basic purification, their procedures often require significant manual labor, and the sample preparation process remains a major source of analytical variability, easily leading to sample loss or quality fluctuations.

[0003] To overcome these challenges, isotachophoresis (ITP) stands out due to its superior focusing capabilities. ITP utilizes a discontinuous buffer system consisting of a leading electrolyte (LE) with high effective mobility and a trailing electrolyte (TE) with lower effective mobility, enabling efficient focusing of target samples (such as nucleic acids) with mobility between the two. This technique can enrich nucleic acids by more than 10,000 times in extremely short times (e.g., less than five minutes) and demonstrates enormous potential for the selective extraction, purification, and quantification of complex biological samples.

[0004] Currently, lab-on-a-chip (ITP) integration has become a research hotspot in the field of laboratory-on-a-chip technology, aiming to reduce human intervention and improve the success rate of generating usable samples from preserved or fresh samples through automated equipment. However, in traditional ITP chip designs, to ensure smooth insertion of external electrodes into the electrolyte reservoir, the opening size of the reservoir is usually designed to be relatively large. This large opening structure significantly increases the contact area between the electrolyte and the external atmosphere, making it easy for gas to seep in or be carried into the microchannels during operation. During electrophoresis, the gas entering the channel forms bubbles, which not only increases the liquid circuit resistance and causes current fluctuations, but in severe cases can even lead to interruption of the electrophoresis circuit and insufficient electrophoretic stability. Summary of the Invention

[0005] The purpose of this invention is to provide a microfluidic chip for isokinetic electrophoresis, which can solve the problem of insufficient stability in isokinetic electrophoresis.

[0006] To solve the above technical problems, embodiments of the present invention provide an isotachophoresis microfluidic chip, comprising: a glass substrate and a polydimethylsiloxane (PDMS) channel bonded to the glass substrate, wherein the PDMS channel comprises, in sequence along the fluid path direction, a trailing electrolyte (TE) electrode region, a main channel region, and a leading electrolyte (LE) electrode region. The PDMS flow channel has openings located above the TE and LE electrode regions. A plunger with a dense PDMS film is fixed to the TE and LE electrode regions by interference fit through the openings. External electrodes are inserted into the TE and LE electrode regions from symmetrical positions on both sides of the PDMS flow channel, with their positions directly opposite the dense PDMS film below the plunger. The plunger is kept under a preset negative pressure after the electrolyte and sample for isotachophoresis are injected into the PDMS flow channel. The external electrodes are inserted into the TE and LE electrode regions after the preset negative pressure is maintained above the plunger. The TE electrode region and LE electrode region on the PDMS flow channel are respectively provided with narrow and long pipe arrays between them and the main flow channel region. Each pipe in the narrow and long pipe array is arranged in parallel and the cross-sectional area is smaller than the cross-sectional area of ​​the main flow channel region.

[0007] Furthermore, the plunger is pre-embedded with a steel needle interface to inject the corresponding electrolyte into the PDMS channel via a hose connected to the steel needle interface above the TE electrode area and the LE electrode area.

[0008] Furthermore, the main channel area includes a pre-enrichment sample inlet and an enrichment sample sampling port, and steel needle interfaces are also pre-embedded on the pre-enrichment sample inlet and the enrichment sample sampling port, respectively, so as to inject and push the sample into the PDMS channel through the pre-enrichment sample inlet and the enrichment sample sampling port through the steel needle interface of the flexible tube, and to remove the sample from the PDMS channel after isokinetic electrophoresis.

[0009] Furthermore, the plunger is a frustum-shaped silicone plunger.

[0010] Furthermore, the external electrode is a Y-shaped electrode.

[0011] Furthermore, the head of the Y-shaped electrode is inserted into the TE electrode region and the LE electrode region, while the tail is exposed outside the isokinetic electrophoresis microfluidic chip to connect to an external power source.

[0012] Furthermore, the Y-shaped electrode is connected to an external power source via alligator clips.

[0013] Furthermore, the PDMS channel is a PDMS block with a channel shape pattern, and the side with the channel shape pattern is bonded to the glass substrate by pressure or thermal bonding.

[0014] The isokinetic electrophoresis microfluidic chip provided by this invention has at least the following beneficial effects: A plunger with a dense PDMS film is installed and fixed in the PDMS channel above the TE and LE electrode regions through an opening. After the external electrode is inserted into the PDMS channel, its position is directly opposite the dense PDMS film below the plunger. Due to the selective permeability of the dense PDMS film, which supports smooth gas exchange, and its hydrophobicity, which prevents liquid overflow, a negative pressure environment is created above the plunger on the dense PDMS film. This allows gas generated during the insertion of the external electrode or the reaction to be expelled, preventing backflow of air. Simultaneously, a long, narrow channel array is arranged between the TE and LE electrode regions and the main channel region in the PDMS channel, serving as a second bubble isolation defense line. This structure utilizes the flow resistance characteristics formed by the long, narrow path to lock any remaining bubbles within the TE and LE electrode regions, preventing them from entering the main channel region and causing resistance fluctuations or open circuits. Therefore, this invention effectively solves the problems of bubble interference and electrical instability, improving the stability of isotachophoresis microfluidic chips performing isotachophoresis.

[0015] Furthermore, this invention transforms the complex thin-film structure into an independent standard part—a plunger with a membrane—which can be directly assembled through an interference fit. This mold structure supports the one-time production of complete mold sets, resulting in lower manufacturing difficulty and cost. Attached Figure Description

[0016] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this invention, illustrate exemplary embodiments of the invention and are used to explain the invention, but do not constitute an undue limitation of the invention. In the drawings:

[0017] Figure 1 A schematic diagram of a conventional isovelocity electrophoresis plate laboratory provided by the present invention; Figure 2 This is a schematic diagram of the region division of an isokinetic electrophoresis microfluidic chip provided by the present invention; Figure 3 A schematic diagram of a microfluidic chip for isokinetic electrophoresis provided by the present invention; Figure 4 This is a schematic diagram illustrating the working principle of an isokinetic electrophoresis microfluidic chip provided by the present invention; Figure 5 This is a schematic diagram showing the design details of an isokinetic electrophoresis microfluidic chip provided by the present invention. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with specific embodiments and corresponding drawings. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0019] Currently, existing on-chip ITP systems still face many challenges in practical application. The limited number of chips available are mostly monopolized by high-end foreign markets, and they generally suffer from complex electrode interface operations, difficult-to-manufacture bubble removal structures, and strong dependence on precision desktop base station equipment. This makes it difficult for grassroots hospitals or laboratories to widely apply related technologies, limiting the application potential of ITP in portable and point-of-care testing (POCT) scenarios.

[0020] like Figure 1 As shown in (a), an existing isotachophoresis chip product includes an ITP channel 1500, in which a driving electrode is disposed in a buffered elution electrode (EH) reservoir 1501 and a buffered lead electrolyte (LEH) reservoir 1502, and a ground electrode is disposed in a buffered trailing electrolyte (TEH) reservoir 1503. The chip also integrates a conductivity detection structure, such as a capacitively coupled non-contact conductivity detector (C4D), whose electrode 1504 can be disposed externally to the chip, such as near the elution reservoir 1505. The channel further includes a lead electrolyte reservoir 1506 and a sample reservoir or injection point 1507. Gas ports (indicated by small circles in the figure) are provided at both ends of the channel, allowing for the automatic loading or introduction of fluid from the attached reservoirs into the channel by applying vacuum or pressure. Figure 1 As shown in (b), the bubble prevention device includes a gas channel or line 1001 for providing the actuation required pneumatic pressure to other areas of the capillary barrier or fluid device; the gas channel 1001 is connected to an external gas pressure source via a pneumatic port 1006. The fluid resistance of the gas channel 1001 is higher than that of the liquid channel, thereby reducing or preventing liquid inflow into the gas channel while providing pressure; for example, the cross-sectional area of ​​the gas channel 1001 is less than half the cross-sectional area of ​​the main isovelocity electrophoresis channel. Multiple gas channels 1001 may be connected to the same gas reservoir or pneumatic port 1006, for example, through a branch channel structure. A capillary barrier 1002 is provided in the gas channel 1001, which is arranged at the liquid channel interface 1003 between the sub-channel connecting the sample 1004 and the lead electrolyte buffer 1005, and can be used in conjunction with branch air lines and capillary valves to prevent upstream liquid from moving toward the pneumatic port 1006, thereby preventing bubbles from entering the fluid channel. Figure 1As shown in (c), the chip requires a tightly fitted benchtop system or base station for isotachophoresis, such as 1300, to perform sample preparation and isotachophoresis on a fluid device cartridge 1301. The fluid device cartridge can be mounted onto the benchtop system 1300. A matching lid and controller 1302 can be lowered and cover the fluid device cartridge for reliable sealing and electrical connection. The benchtop system also includes a control panel 1303 (such as a touchscreen) with a user interface for system operation and parameter settings. The system integrates a pressure control module and can receive pressure feedback signals to regulate the loading, transport, and... Injection: Fluid can be introduced into the drying channel on the device. Pressure regulation can be achieved through a solenoid valve. The benchtop system also integrates electrodes or electrical contacts to electrically connect to the reservoir or contacts on the fluid device box 1301, thereby applying an electric field inside the device. It can be configured with various detection units such as photodetectors, reflection sensors, infrared detectors, electrical detectors, thermal sensors, flow sensors, and pressure sensors. Among them, photodetectors include CMOS, CCD, photodiode, and other types. Electrical detectors can track the position of nucleic acid bands by monitoring the change in conductivity at the interface between the leading electrolyte and the trailing electrolyte. Thermal sensors include infrared sensors, thermistors, RTDs, and thermocouples. The entire device is very expensive.

[0021] Therefore, existing isokinetic electrophoresis microfluidic chips have the following problems: 1. Bubble interference and electrical instability caused by electrode interface design: In traditional isotachyplasty (ITP) chip designs, a relatively large opening size is typically required to ensure smooth insertion of the external electrode 1504 into the electrolyte reservoir (e.g., 1501, 1502, 1503). However, this large opening significantly increases the contact area between the electrolyte and the external atmosphere, making it easy for gas to seep in or be carried into the microchannels during operation. During electrophoresis, the gas entering the channel forms bubbles, which not only increases the liquid circuit resistance and causes current fluctuations, but can also, in severe cases, lead to interruption of the electrophoresis circuit, thus affecting the repeatability and accuracy of the detection results.

[0022] 2. The complex flow path control structure leads to higher manufacturing and operating costs: To achieve gas exchange and liquid blockage at the flow channel interface (e.g., at 1003), existing chips typically require the integration of numerous capillary barriers 1002 or complex air pipelines. This design demands high precision in material processing, increasing chip production costs. Furthermore, the system relies on a precision pneumatic pump drive and branch channel structure for timing control. Due to the large number of mechanical components involved and frequent gas path switching, imbalances or leaks can easily occur during actual pressure regulation, making it difficult to fully guarantee the operational stability of different batches of chips.

[0023] 3. High dependence on supporting equipment: like Figure 1 As shown in (c), the operation of existing chips typically relies on a complex desktop base station system 1300. This system integrates a pressure control module, a precision cover plate mechanism 1302, and a sensor array encompassing various types, including optical, electrical, and thermal sensors. This reliance on high-precision supporting equipment results in a high overall cost per test, a large system size, and relatively inconvenient maintenance. For applications seeking low cost, portability, and point-of-care testing (POCT), this complex system architecture raises the barrier to entry and limits the widespread adoption of microfluidic chips in grassroots environments.

[0024] Therefore, there is a need to develop a chip that can achieve stable sample loading and ITP processes, while also automatically removing air bubbles, facilitating assembly and production, and simplifying operation. The core objective of this invention is to overcome this challenge by optimizing the chip structure and operating mechanism to provide a novel, low-barrier-to-entry, and highly stable technical solution.

[0025] To address the three problems currently existing in isokinetic electrophoresis microfluidic chips, this invention aims to solve the following three interrelated key technical issues: how to optimize the electrode interface coupling method to synergistically suppress the interference of bubbles on electrophoretic stability; how to reduce the manufacturing complexity and cost of the chip through the simplification of the flow control mechanism and modular construction; and how to reduce the dependence on supporting equipment for the microfluidic system.

[0026] The technical solutions provided by the various embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0027] One embodiment of the present invention relates to an isotachophoresis microfluidic chip. The implementation details of the isotachophoresis microfluidic chip of this embodiment are described in detail below. The following implementation details are provided for ease of understanding and are not necessary for implementing this solution.

[0028] The specific structure of the isokinetic electrophoresis microfluidic chip in this embodiment can be as follows: Figure 2 and Figure 3 As shown, it includes: a glass substrate and polydimethylsiloxane (PDMS) channels bonded to the glass substrate. The PDMS channels sequentially include a trailing electrolyte (TE) electrode region, a main channel region, and a leading electrolyte (LE) electrode region along the fluid path direction. The PDMS channels are PDMS blocks with a channel shape pattern, and the side with the channel shape pattern is bonded to the glass substrate by pressure or thermal bonding.

[0029] The PDMS flow channel has openings located above the TE and LE electrode regions. A plunger with a dense PDMS film is fixed to the TE and LE electrode regions by interference fit through the openings. External electrodes are inserted into the TE and LE electrode regions from symmetrical positions on both sides of the PDMS flow channel, with their positions directly opposite the dense PDMS film below the plunger. The plunger is kept under a preset negative pressure after the electrolyte and sample for isotachophoresis are injected into the PDMS flow channel. The external electrodes are inserted into the TE and LE electrode regions after the preset negative pressure is maintained above the plunger.

[0030] In one example, the plunger in this embodiment has a pre-embedded steel needle interface to inject the corresponding electrolyte into the PDMS channel via a flexible tube connected to the steel needle interface above the TE and LE electrode areas. The main channel area includes a pre-enrichment sample inlet and an enrichment sample sampling port, both of which also have pre-embedded steel needle interfaces to inject samples into the PDMS channel via a flexible tube connected to the steel needle interface, and to remove samples from the PDMS channel after isotachophoresis. The plunger is a frustoconical silicone stopper.

[0031] In one example, the external electrode in this embodiment is a Y-shaped electrode. The head of the Y-shaped electrode is inserted into the TE electrode region and the LE electrode region, while the tail is exposed outside the isovelocity electrophoresis microfluidic chip for connection to an external power source. For example, the external power source can be connected via alligator clips.

[0032] The TE electrode region and LE electrode region on the PDMS flow channel are respectively provided with narrow and long pipe arrays between them and the main flow channel region. Each pipe in the narrow and long pipe array is arranged in parallel and the cross-sectional area is smaller than the cross-sectional area of ​​the main flow channel region.

[0033] The isotachophoresis microfluidic chip of the present invention is a device that can be used for isotachophoresis processes, automatic sample introduction, and bubble removal. See [link to relevant documentation]. Figure 2 The chip is made of materials such as glass substrate, PDMS channel, plunger, and steel needle. According to the role of each component in the automated isovelocity electrophoresis process, the channel can be marked from left to right as TE (tailing electrolyte) electrode area, bubble isolation structure (narrow channel or salt bridge), pre-enrichment sample inlet, electrolyte interface (pore), enrichment sample sampling port, and LE (leading electrolyte) electrode area.

[0034] See details Figure 3The bottom of the isotachophoresis microfluidic chip is a glass substrate, which serves as the chip's base. The flow channel refers to a PDMS block with a flow channel shape. The patterned side is bonded to the glass substrate by pressure or thermal bonding, forming a semi-enclosed space for liquid to pass through. This PDMS block is connected to other accessories through perforation. Four steel needles are pre-embedded in the entire flow channel (closed when not connected to the tubing) to inject reagents or RNA samples into the flow channel while reducing the generation of air bubbles. Symmetrical holes are opened on the side of the PDMS block. After the solution and reagents are injected into the flow channel, electrodes are inserted from both sides. In order to eliminate air bubbles generated during the ITP process or when inserting electrodes, a large hole is designed above the LE and TE electrode areas. A frustum-shaped silicone plug or PDMS plunger with a dense PDMS film is interference-fitted. The dense PDMS film allows gas to pass through while blocking liquid.

[0035] As can be seen, this invention designs a symmetrical insertable Y-type gold electrode structure adapted to alligator clip connections: it employs an insertable Y-type gold electrode and features a symmetrical layout design based on the long end of the flow channel (e.g., Figure 3 (As shown). This symmetrical distribution helps to create a uniform and stable electric field environment within the microchannels, thereby improving the consistency of electrophoretic separation. The electrodes are made of gold, designed to leverage its excellent chemical stability and effectively prevent reactions with corrosive chemical reagents during electrophoresis. For electrical connections, the tail of the Y-shaped electrode is exposed outside the chip, allowing for a secure connection to an external power source using only standard alligator clips (e.g., ...). Figure 5 As shown), this significantly simplifies the wiring process. Furthermore, the electrode insertion position is directly aligned with the underlying dense PDMS film (as shown). Figure 4 As shown in (b), this layout is beneficial for eliminating bubbles generated during electrode insertion or reaction in real time, ensuring the continuity of the electrical circuit.

[0036] Simultaneously, a narrow flow channel array with bubble interception function and its physical barrier design were implemented: to prevent bubbles from entering the main loop flow channel, a narrow gas isolation channel (such as...) was set between the electrode area and the main flow channel. Figure 4 (as shown in (c)). This structure utilizes the flow resistance characteristics formed by the narrow path to reliably intercept bubbles within the electrode area, preventing them from interfering with the detection area. Furthermore, the simple geometry of the channel reduces the complexity of the processing mold. Considering that a single channel may significantly affect the liquid path resistance, this invention further optimizes it into a flow channel array structure. By precisely controlling the array spacing, this design maintains effective gas isolation while minimizing resistance to ion migration and fluid flow, ensuring the electric field strength and flow field efficiency during electrophoresis.

[0037] Additionally, a modular plunger packaging structure integrating steel pins and PDMS film: employs a silicone plug or plunger assembly with a dense PDMS film, and integrates a standardized steel pin interface (such as...). Figure 4 (As shown in (d)). The thickness of the PDMS film is precisely controlled, utilizing its unique selective permeability to support smooth gas exchange while preventing liquid leakage through hydrophobicity. The steel needles within the component not only serve as convenient access points for external driving devices (such as syringes) but also promptly expel air from the connecting tubing during the initial injection of chemical reagents. In terms of manufacturing processes, this plug-type design replaces the traditional method of directly processing thin films on the chip substrate, allowing complex thin film structures to be modularly assembled as independent components. Simultaneously, this structure allows for the one-time production of complete molds during the soft lithography stage, significantly simplifying the mold structure and improving production and assembly efficiency.

[0038] Based on the above structure, the usage steps of the isokinetic electrophoresis microfluidic chip of the present invention are as follows: Figure 5 As shown, it includes: ① Inject reagents, connect the appropriate tubing to the steel needles reserved above the LE and TE electrode areas, and use an external precision volume control device (precision syringe, injection pump or peristaltic pump) to push the corresponding electrolyte into the flow channel. The electrolyte interface is open to the atmosphere, so maintaining a reasonable on / off sequence can control the interface between LE and TE at the reserved position. ② After LE and TE injections are completed, seal both ends of the steel needles, and then use the same external device to inject the sample to be enriched from the sample injection port (e.g., Figure 2 (as shown in the image) ③ After all reagents have entered the flow channel, a certain negative pressure is maintained above the PDMS membrane until the ITP process is completed, and the designed Y-type electrode is inserted from both sides. ④ Use alligator clips to hold the electrode, connect the other end to a power source (e.g., Keithley 2410), and set the appropriate parameters to start electrophoresis.

[0039] Use appropriate methods to determine when the enriched sample band has reached the designated position, and use a syringe, syringe pump or pipette to remove the sample from the sampling port for downstream operations.

[0040] In summary, this invention provides a de-integrated portable isotachophoresis chip. By employing symmetrically distributed insertable Y-shaped gold electrodes, a modular plunger assembly integrating PDMS film and steel needles, and a narrow flow channel array with bubble interception capabilities, it effectively solves the problems of complex electrode interfaces, severe bubble interference, and high dependence on expensive supporting equipment in existing technologies. This design not only significantly reduces manufacturing and consumable costs through modular assembly but also allows users to achieve stable electrophoresis operations and sample recovery using conventional equipment such as alligator clips and syringes, significantly improving the accessibility of microfluidic systems in primary healthcare and on-site testing scenarios.

[0041] Specifically, the beneficial effects of the isokinetic electrophoresis microfluidic chip of the present invention are as follows: (1) Significantly improves the stability and reproducibility of the electrophoresis process: Through the synergistic effect of the symmetrically distributed Y-shaped gold electrodes and the PDMS dense film, the system can eliminate bubbles generated by electrode insertion and electrolysis reaction in real time. Combined with the gas isolation channel in the form of a flow channel array, the bubbles are effectively intercepted outside the electrode area, avoiding current fluctuations or circuit interruptions caused by bubbles entering the main circuit, and ensuring the steady state of the electric field environment.

[0042] (2) Significantly reduced manufacturing complexity and production costs: By adopting a "component-based" assembly approach, complex thin film structures are transformed into standardized silicone plugs or plunger assemblies. This modular design simplifies the structure of soft lithography molds, supports one-time production of complete mold sets, avoids the high-difficulty process of directly processing heterogeneous thin films on chip substrates, and significantly improves the yield of large-scale mass production.

[0043] (3) It achieves extremely low operating threshold and high portability: The chip interface design realizes "de-integration", and electrical connection can be completed using a common alligator clip without the need for expensive special electrode sockets. At the same time, through the plug structure with integrated steel needle, users only need to use conventional syringes or pipettes and other basic laboratory equipment to complete sample injection, venting and sampling, completely getting rid of the dependence on large and complex desktop base station systems, which is extremely conducive to promotion to grassroots hospitals and on-site testing scenarios.

[0044] (4) Enhanced chemical resistance and structural reliability: The use of gold electrodes effectively solves the risk of reaction between electrodes and chemical reagents, and extends the storage and service life of consumables. In addition, the flow channel array design ensures the bubble interception function while reducing the flow path resistance through multi-channel parallel connection, thus balancing the fluid dynamics performance and electrical performance.

[0045] Those skilled in the art will understand that the above embodiments are specific embodiments for implementing the present invention, and in practical applications, various changes in form and detail can be made without departing from the spirit and scope of the embodiments of the present invention. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the embodiments of the present invention; therefore, the scope of protection of the embodiments of the present invention should be determined by the scope defined in the claims.

Claims

1. A microfluidic chip for isokinetic electrophoresis, characterized in that, include: The glass substrate and the polydimethylsiloxane (PDMS) flow channel bonded to the glass substrate, wherein the PDMS flow channel includes, in sequence along the fluid path direction, a trailing electrolyte (TE) electrode region, a main flow channel region, and a leading electrolyte (LE) electrode region. The PDMS flow channel has openings located above the TE and LE electrode regions. A plunger with a dense PDMS film is fixed to the TE and LE electrode regions by interference fit through the openings. External electrodes are inserted into the TE and LE electrode regions from symmetrical positions on both sides of the PDMS flow channel, with their positions directly opposite the dense PDMS film below the plunger. The plunger is kept under a preset negative pressure after the electrolyte and sample for isotachophoresis are injected into the PDMS flow channel. The external electrodes are inserted into the TE and LE electrode regions after the preset negative pressure is maintained above the plunger. The TE electrode region and LE electrode region on the PDMS flow channel are respectively provided with narrow and long pipe arrays between them and the main flow channel region. Each pipe in the narrow and long pipe array is arranged in parallel and the cross-sectional area is smaller than the cross-sectional area of ​​the main flow channel region.

2. The isokinetic electrophoresis microfluidic chip according to claim 1, characterized in that, The plunger has a pre-embedded steel needle interface, which is used to inject the corresponding electrolyte into the PDMS channel through a hose connected to the steel needle interface above the TE electrode area and the LE electrode area.

3. The isokinetic electrophoresis microfluidic chip according to claim 2, characterized in that, The main channel includes a pre-enrichment sample inlet and an enrichment sample sampling outlet. Steel needle interfaces are also pre-embedded on the pre-enrichment sample inlet and the enrichment sample sampling outlet, respectively, so that samples can be injected into the PDMS channel through the steel needle interfaces connected by a flexible tube, and samples can be removed from the PDMS channel after isokinetic electrophoresis.

4. The isokinetic electrophoresis microfluidic chip according to claim 1, characterized in that, The plunger is a frustum-shaped silicone plunger.

5. The isokinetic electrophoresis microfluidic chip according to claim 1, characterized in that, The external electrode is a Y-shaped electrode.

6. The isokinetic electrophoresis microfluidic chip according to claim 5, characterized in that, The head of the Y-shaped electrode is inserted into the TE electrode region and the LE electrode region, while the tail is exposed outside the isokinetic electrophoresis microfluidic chip for connection to an external power source.

7. The isokinetic electrophoresis microfluidic chip according to claim 6, characterized in that, The Y-shaped electrode is connected to an external power source via alligator clips.

8. The isokinetic electrophoresis microfluidic chip according to claim 1, characterized in that, The PDMS channel is a PDMS block with a channel shape pattern, and the side with the channel shape pattern is bonded to the glass substrate by pressure or thermal bonding.

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