Electrowetting micro-fluidic card box

Abstract

The invention discloses an electrowetting microfluidic card box, and relates to the technical field of electrowetting microfluidic control, an upper electrode plate comprises an upper substrate, an upper conductive layer and an upper hydrophobic layer, and an upper insulating layer is arranged in at least a local area between the upper conductive layer and the upper hydrophobic layer; the lower polar plate comprises a lower substrate, a lower insulating layer and a lower hydrophobic layer, and a driving electrode is arranged on the lower substrate and drives liquid to move in the liquid drop channel; in the area where the upper insulating layer is arranged, the upper portions of the liquid drops make contact with the upper hydrophobic layer and are insulated from the upper conductive layer, charges of the upper conductive layer cannot be conducted to the liquid drops, meanwhile, the lower portions of the liquid drops make contact with the lower hydrophobic layer and are insulated through the lower insulating layer, the liquid drops are made to be high in overall height, the three-dimensional performance of the liquid drops is higher, and the liquid drops are easier to drive; in addition, due to the fact that the liquid drops make contact with the upper hydrophobic layer more tightly, the contact area of the liquid drops and the upper hydrophobic layer is larger, and optical detection of the liquid drops is more accurate.

Description

Technical Field

[0001] The present invention relates to the technical field of electrowetting microfluidic control, and further to an electrowetting microfluidic cartridge. Background Art

[0002] Closed microfluidic cartridges that integrate sample preparation, nucleic acid purification, and PCR (polymerase chain reaction) amplification detection are currently a major development direction in molecular diagnostics. With increasing demands for product performance, achieving rapid, highly sensitive detection of multiplexed nucleic acids in fully integrated microfluidic cartridges has become a key goal.

[0003] At present, the main factor restricting the detection speed is the temperature rise and fall rate during the PCR amplification process, and the key to achieving high-sensitivity detection lies in the precise control of the reagent reaction process.

[0004] Electrowetting technology eliminates the need to control the heating and cooling of a specific area. Instead, it simply sets different temperature zones, allowing droplets to transfer between them, thereby heating and cooling the droplets. Due to its precise and flexible control of tiny droplets, electrowetting technology can rapidly switch droplets between different PCR temperature zones, achieving rapid temperature changes and significantly shortening PCR reaction times, making it an effective method for rapid nucleic acid detection.

[0005] However, during the PCR reaction, the electric field introduced by the electrowetting system and the thermal field required by the PCR reaction itself will be superimposed, which will cause serious bubble problems. The more bubbles there are, the more likely it is to cause droplet drive failure and inaccurate reaction fluorescence signal collection.

[0006] Furthermore, to further enhance the sensitivity and detection rate of nucleic acid testing, the market typically increases sample volume during sample preparation and nucleic acid purification, thereby obtaining more target nucleic acids for PCR amplification testing. However, traditional electrowetting systems, with their strengths in controlling small droplets, struggle to quickly and stably manipulate large droplets larger than 20 μL, and are even less compatible with sample preparation and nucleic acid purification processes involving volumes exceeding several hundred microliters.

[0007] Therefore, traditional electrowetting PCR will produce serious bubble problems, affecting the driving stability and the accuracy of fluorescence signal acquisition; and traditional electrowetting cannot manipulate large-volume droplets. Summary of the Invention

[0008] The core of the present invention is to provide an electrowetting microfluidic cartridge. By providing an upper insulating layer in at least a partial area between the upper conductive layer and the upper hydrophobic layer, the droplets are electrically neutral in the area covered by the upper insulating layer. The droplets maintain a more three-dimensional shape, enabling large droplet control, reducing bubble generation, and achieving more accurate photometry. The specific solution is as follows:

[0009] An electrowetting microfluidic cartridge includes a reagent reaction module, wherein the reagent reaction module includes an upper electrode plate and a lower electrode plate that are sealed and assembled with each other, and a droplet channel is formed between the upper electrode plate and the lower electrode plate;

[0010] The upper electrode plate includes an upper substrate, an upper conductive layer, an upper insulating layer and an upper hydrophobic layer;

[0011] The upper insulating layer is provided at least partially between the upper conductive layer and the upper hydrophobic layer; the thickness of the upper insulating layer is 0.1 μm-20 μm;

[0012] The lower electrode plate includes a lower substrate, a lower insulating layer and a lower hydrophobic layer, and the lower substrate is provided with a driving electrode.

[0013] Optionally, the reagent reaction module is provided with a reagent distribution area, a nucleic acid purification area, a nucleic acid amplification area and a waste liquid area;

[0014] The upper insulating layer is arranged in the nucleic acid purification area and / or the nucleic acid amplification area.

[0015] Optionally, the nucleic acid amplification area is provided with a plurality of nucleic acid amplification flow channels distributed side by side, and each of the nucleic acid amplification flow channels is used for flow of droplets;

[0016] The upper insulating layer is provided in the corresponding area of ​​each nucleic acid amplification channel.

[0017] Optionally, at least two different temperature zones are provided in the region corresponding to the nucleic acid amplification flow channel, and the droplets move back and forth in the nucleic acid amplification flow channel to transfer between different temperature zones.

[0018] Optionally, the temperature region includes a high temperature zone not lower than 60°C.

[0019] Optionally, an optical signal collection area is provided in the region corresponding to the nucleic acid amplification flow channel for optical equipment to collect signals.

[0020] Optionally, bubble capture structures are provided on both sides of the nucleic acid amplification channel.

[0021] Optionally, a reagent storage module is further included, and the reagent storage module is located above the reagent dispensing area;

[0022] The reagent storage module comprises a frame and a top cover assembled with each other, the frame is provided with a plurality of storage cavities, and the bottom end of the storage cavity is provided with an aluminum-plastic film;

[0023] A flexible film is provided on the top cover, and the upper end of at least part of the storage cavity is correspondingly provided with the flexible film; the storage cavity for storing samples is provided with a sample plug.

[0024] Optionally, the upper substrate is provided with a puncture column for puncturing the aluminum-plastic film provided at the bottom end of the storage cavity when the reagent storage module is pressed downward.

[0025] Optionally, the top cover is provided with an air hole, and an air hole plug is provided at the upper end of the air hole; the air hole plug is used to seal the air hole after the reagent storage module is pressed down and punctured.

[0026] The present invention provides an electrowetting microfluidic cartridge, wherein a reagent reaction module comprises an upper electrode plate and a lower electrode plate which are sealed and assembled with each other, and a droplet channel for droplet circulation is formed between the upper electrode plate and the lower electrode plate; the upper electrode plate comprises an upper substrate, an upper conductive layer and an upper hydrophobic layer, and an upper insulating layer is provided at least in a local area between the upper conductive layer and the upper hydrophobic layer; the lower electrode plate comprises a lower substrate, a lower insulating layer and a lower hydrophobic layer, and a driving electrode is provided on the lower substrate to drive the liquid to move in the droplet channel; since the upper insulating layer is provided in a local area between the upper conductive layer and the upper hydrophobic layer, the upper part of the droplet contacts the upper insulating layer in the area where the upper insulating layer is provided The hydrophobic layer is insulated from the upper conductive layer, and the charge of the upper conductive layer will not be conducted to the droplet. At the same time, the lower part of the droplet contacts the lower hydrophobic layer and is insulated by the lower insulating layer. The droplet remains electrically neutral in the area with the upper insulating layer, and forms a three-dimensional shape under the action of surface tension and electric field, so that the droplet as a whole maintains a higher height, the droplet is easier to be driven, and the droplet is not easy to break. Due to the higher three-dimensionality of the droplet, it is not easy to generate bubbles during driving. In addition, because the droplet is in closer contact with the upper hydrophobic layer, the droplet has a larger contact area with the upper hydrophobic layer, and the optical detection of the droplet is more accurate. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0028] Figure 1 Schematic diagram of the overall structure of the electrowetting microfluidic cartridge of the present invention;

[0029] Figure 2 A diagram showing the relative relationship between the reagent reaction module and the reagent storage module;

[0030] Figure 3 This is a schematic diagram of the area division of the reagent reaction module;

[0031] Figure 4 It is the relative relationship diagram of the upper plate and the lower plate;

[0032] Figure 5 This is the exploded view of the upper plate;

[0033] Figure 6 This is the exploded view of the lower plate;

[0034] Figure 7 This is an exploded view of the reagent storage module;

[0035] Figure 8A This is the front view of the liquid droplet morphology on the upper plate without the insulating layer;

[0036] Figure 8B Schematic diagram of the top plate without insulating layer and large volume droplet driven tearing;

[0037] Figure 9A This is a front view of the droplet shape with an insulating layer on the upper plate;

[0038] Figure 9B Schematic diagram of the top plate with an insulating layer and a large volume droplet driven normally from a top view;

[0039] Figure 10A This is a front view schematic diagram of the upper plate without an insulating layer - droplet driving form and bubble generation;

[0040] Figure 10B This is a top view of the upper plate without an insulating layer - droplet driving form and bubble generation;

[0041] Figure 11A It is a front view schematic diagram of the upper electrode plate with an insulating layer - the droplet driving form and bubble generation;

[0042] Figure 11B The top view of the insulating layer on the upper plate - droplet driving form and bubble generation;

[0043] Figure 12A Schematic diagram of the top plate without insulation layer and the droplet blocked by the bubble;

[0044] Figure 12B This is a top view of the upper plate without an insulating layer - the droplet is blocked by bubbles;

[0045] Figure 13 Schematic diagram of the optical measurement of four different droplet morphologies;

[0046] Figure 14 A top view of the bubble trapping structure set up in the nucleic acid amplification area.

[0047] The diagram includes:

[0048] Reagent reaction module 10, reagent distribution area 101, nucleic acid purification area 102, nucleic acid amplification area 103, nucleic acid amplification flow channel 1031, bubble capture structure 1032, waste liquid area 104;

[0049] Upper electrode plate 110, upper substrate 111, piercing pillar 1111, upper conductive layer 112, upper insulating layer 113, upper hydrophobic layer 114, lower electrode plate 120, lower substrate 121, lower insulating layer 122, lower hydrophobic layer 123, driving electrode 124, sealing ring 130;

[0050] Reagent storage module 20 , frame 210 , storage cavity 211 , aluminum-plastic film 212 , sample plug 213 , top cover 220 , flexible film 221 , air hole 222 , air hole plug 223 . DETAILED DESCRIPTION

[0051] In order to enable those skilled in the art to better understand the technical solution of the present invention, the electrowetting microfluidic cartridge of the present invention will be described in detail below with reference to the accompanying drawings and specific implementation methods.

[0052] The present invention provides an electrowetting microfluidic cartridge, combined with Figure 1 、 Figure 2 As shown, the electrowetting microfluidic cartridge includes a reagent reaction module 10, which is used to realize liquid drive control.

[0053] Electrowetting microfluidics eliminates the need for pumps, valves, and pipelines. Instead, it applies an external electric field to alter the solid-liquid contact angle, causing asymmetric droplet deformation and enabling precise manipulation of droplets. Because the input voltage driving the electrode array can be controlled using flexible digital programming, it is also known as digital microfluidics.

[0054] Combine Figure 4 As shown, the reagent reaction module 10 includes an upper plate 110 and a lower plate 120 that are sealed against each other. A sealing ring 130 is provided between the upper plate 110 and the lower plate 120 to keep the gap between the upper plate 110 and the lower plate 120 sealed. A droplet channel is formed between the upper plate 110 and the lower plate 120, and droplets can move in the gap between the upper plate 110 and the lower plate 120. Specifically, the upper hydrophobic layer 114 of the upper plate 110 contacts the droplets, and the lower hydrophobic layer 123 of the lower plate 120 contacts the droplets.

[0055] Combine Figure 5As shown, the upper electrode plate 110 includes an upper substrate 111, an upper conductive layer 112, an upper insulating layer 113, and an upper hydrophobic layer 114. The upper substrate 111, the upper conductive layer 112, the upper insulating layer 113, and the upper hydrophobic layer 114 are arranged in a stacked relationship, from top to bottom. The upper insulating layer 113 acts as an insulator between the upper conductive layer 112 and the upper hydrophobic layer 114.

[0056] An upper insulating layer 113 is disposed in at least a portion of the area between the upper conductive layer 112 and the upper hydrophobic layer 114. That is, the upper insulating layer 113 does not need to cover the entire area of ​​the upper electrode plate 110; the upper insulating layer 113 only needs to be disposed in a portion of the area. Of course, if desired, the present invention allows the upper insulating layer 113 to be disposed in all of the aforementioned areas.

[0057] Combine Figure 6 As shown, in order from bottom to top, the lower electrode plate 120 includes a lower substrate 121, a lower insulating layer 122, and a lower hydrophobic layer 123. The lower substrate 121 is provided with a driving electrode 124. The driving electrode 124 is formed by a plurality of electrode blocks. The electrode blocks are arranged and distributed on the lower substrate 121 according to a preset pattern. The driving electrode 124 is used to apply a driving force to the droplet, causing the droplet to move between the upper hydrophobic layer 114 and the lower hydrophobic layer 123. The area of ​​the lower insulating layer 122 and the lower substrate 121 is roughly equal. The lower insulating layer 122 insulates every area of ​​the lower electrode plate 120, and the droplet does not conduct electricity with the driving electrode 124 no matter where it moves.

[0058] In the present invention, an upper insulating layer 113 is provided in a local area of ​​the upper electrode plate 110 to insulate the upper conductive layer 112. The lower insulating layer 122 insulates the driving electrode 124. The upper conductive layer 112 and the driving electrode 124 are respectively connected to different electrodes.

[0059] If the upper electrode plate 110 is not provided with the upper insulating layer 113, or in the area where the upper insulating layer 113 is not provided, the upper conductive layer 112 forms a conductive contact with the droplet, causing the droplet to be charged. This charge is opposite to the charge polarity of the driving electrode 124. Opposite charges attract each other, and the driving electrode 124 attracts the droplet, causing the droplet to move closer to the lower hydrophobic layer 123, and the droplet forms a shape that is small at the top and large at the bottom (such as Figure 8A As shown in FIG, the area of ​​the droplet contacting the lower hydrophobic layer 123 is much larger than the area of ​​the droplet contacting the upper hydrophobic layer 114. Since the droplet has a large contact area with the lower hydrophobic layer 123 and is attracted by the driving electrode 124, when the droplet is driven by the driving electrode 124, the droplet is easily broken and it is difficult to maintain the shape of a single droplet. Figure 8BThe figure shows the movement of a large 650 μL droplet. An image is captured every 0.6 seconds, with images taken at 0.6 seconds, 1.2 seconds, 1.8 seconds, 2.4 seconds, 3.0 seconds, and 3.6 seconds of the droplet's motion, demonstrating the droplet breakage process. Without the upper insulating layer 113, only small droplets can be driven. Large droplets, due to their large size, have low three-dimensionality and are spread flat on the lower hydrophobic layer 123. The contact area between the droplet and the lower hydrophobic layer 123 is larger, making it difficult to synchronously drive the different positions of the droplet. Maintaining the shape of a single droplet during motion makes it more difficult, making droplet breakage more likely to occur.

[0060] In the present invention, an upper insulating layer 113 is provided between the upper conductive layer 112 and the upper hydrophobic layer 114. The upper insulating layer 113 forms electrical insulation for the upper conductive layer 112. The upper end of the droplet is no longer charged by the upper conductive layer 112. At the same time, the lower end of the droplet is insulated from the driving electrode 124 by the lower insulating layer 122. The droplet is almost unaffected by the charge of the upper conductive layer 112 and the driving electrode 124 and maintains electrical neutrality. Under the condition of electrical neutrality, the droplet presents a full three-dimensional shape (such as Figure 9A As shown in the figure, the contact state between the droplet and the upper hydrophobic layer 114 and the contact state between the droplet and the lower hydrophobic layer 123 are almost the same. The area of ​​the droplet contacting the upper hydrophobic layer 114 is roughly equal to the area of ​​the droplet contacting the lower hydrophobic layer 123. The area of ​​the droplet contacting the lower hydrophobic layer 123 is smaller, and when the droplet is driven to move, the droplet is less likely to break. Therefore, in the area where the upper insulating layer 113 is set, it is possible to drive large-volume droplets. Figure 9B As shown in the figure, the movement of a large volume droplet of 650 μL is demonstrated. An image is acquired every 0.6 s, and images are taken at 0.6 s, 1.2 s, 1.8 s, 2.4 s, 3.0 s, and 3.6 s of the droplet movement. During the entire movement of the droplet, there is no droplet breakage, and the droplet is always kept intact.

[0061] Combine Figure 10A and Figure 10B The figure shows the droplet driving morphology and bubble generation when the upper electrode plate 110 has no upper insulating layer 113. When the droplet is in a flat shape, the droplet is more attached to the lower hydrophobic layer 123, and the electric field causes the Taylor cone effect on the upper end of the droplet, resulting in bubble problems.

[0062] Combine Figure 11A and Figure 11B Figure 1 shows the droplet drive configuration and bubble generation when the upper insulating layer 113 is provided on the upper electrode plate 110. When the droplet is in a three-dimensional configuration, the contact area between the upper end of the droplet and the upper hydrophobic layer 114 is larger, reducing the Taylor cone effect caused by the electric field on the upper end of the droplet, thereby significantly reducing the number of bubbles generated during the PCR process.

[0063] Combine Figure 12A and Figure 12B As shown, Figure 12A (A) (B) (C) show the process of droplets being blocked by bubbles. Flat droplets are more likely to be mixed with gas during movement and more likely to break (e.g. Figure 12B As shown in FIG. 1 ). The present invention provides an upper insulating layer 113 on the upper electrode plate 110. For small amounts of bubbles generated, the three-dimensional droplets can more easily push these bubbles into the bubble capture structures on both sides (details will be described later), thus preventing the droplets from being blocked by bubbles and causing driving failure or being cut off by bubbles.

[0064] At the same time, the three-dimensional droplet morphology has a higher stability in fluorescence signal acquisition compared to the traditional electrowetting flat morphology. Figure 13 As shown in the figure, four different droplet states, ABCD, are respectively shown for optical measurement. Three of them, ABC, represent droplet states without an upper insulating layer 113 on the upper plate 110. Because the upper end of the droplet cannot adhere closely to the upper hydrophobic layer 114, a gap or bubble is formed, making it more difficult for the detection light to penetrate the interior of the droplet, making high-precision detection difficult. D shows a droplet state with an upper insulating layer 113 on the upper plate 110. Because the droplet is more three-dimensional, the upper end of the droplet can adhere closely to the upper hydrophobic layer 114, allowing the detection light to better illuminate the interior of the droplet, making it easier to achieve accurate optical signal acquisition.

[0065] It can be seen that the present invention can produce three major technical effects by providing an upper insulating layer 113 between the upper conductive layer 112 and the upper hydrophobic layer 114: 1) driving droplets of larger volumes; 2) reducing the generation of bubbles in the droplets; and 3) making optical detection more accurate.

[0066] like Figure 3 As shown, it is equivalent to a top view; the entire reagent reaction module 10 is provided with a reagent distribution area 101, a nucleic acid purification area 102, a nucleic acid amplification area 103 and a waste liquid area 104, and different areas are divided according to their functions. Figure 3 In the top-down observation direction, the different areas are equivalent to different parts divided in the horizontal plane, and there is an adjacent relationship between the different areas.

[0067] The reagent distribution area 101 is used to manipulate samples and liquid reagents to achieve precise quantitative distribution, and then enter the nucleic acid purification area 102; the nucleic acid purification area 102 is pre-embedded with dry magnetic beads, and cooperates with liquid reagents to complete the nucleic acid purification process. After the purified nucleic acid is mixed with PCR buffer, several PCR pre-reaction droplets are further quantitatively separated through the reagent distribution area 101 and enter the nucleic acid amplification area 103.

[0068] The upper substrate 111, the upper conductive layer 112 and the upper hydrophobic layer 114 are arranged on the entire area of ​​the upper electrode plate 110. Figure 5 As shown, the upper substrate 111, upper conductive layer 112, and upper hydrophobic layer 114 have approximately equal projected areas on a horizontal plane, respectively covering the aforementioned reagent dispensing area 101, nucleic acid purification area 102, nucleic acid amplification area 103, and waste liquid area 104. The sealing area lacks a hydrophobic layer (as this affects the seal). Other non-droplet manipulation areas, such as the waste liquid area 104, bubble capture structure 1032, and the sealing area, may be free of conductive and hydrophobic layers.

[0069] On the basis of the above scheme, combined with Figure 3 、 Figure 5 As shown, the upper insulating layer 113 is provided in the nucleic acid purification area 102 and the nucleic acid amplification area 103, but is not provided in the reagent dispensing area 101 and the waste liquid area 104. The provision of the upper insulating layer 113 helps to enhance the three-dimensionality of the droplets, allowing for the propulsion of larger droplets while making it less likely for the droplets to break and separate. However, the reagent dispensing area 101 requires quantitative removal of a certain volume of droplets, so the upper insulating layer 113 is not required in the reagent dispensing area 101, ensuring that the reagent dispensing area 101 can accurately extract droplets.

[0070] In the nucleic acid purification area 102 , the sample is adsorbed by magnetic beads to achieve nucleic acid aggregation, and other liquids are discharged into the waste liquid area 104 . The waste liquid area 104 has no requirements on the shape of the droplets, so the upper insulating layer 113 is not required.

[0071] The nucleic acid amplification area 103 is provided with a plurality of nucleic acid amplification flow channels 1031 arranged side by side, each nucleic acid amplification flow channel 1031 is used for the flow of droplets; Figure 3 As shown, there are 8 nucleic acid amplification channels 1031, each of which can accommodate a droplet of liquid to move back and forth, and the moving direction is shown by the double-headed arrow. The corresponding area of ​​each nucleic acid amplification channel 1031 is provided with an upper insulating layer 113, as shown in FIG. Figure 5 As shown, each nucleic acid amplification flow channel 1031 is provided with an upper insulating layer 113. To facilitate assembly, one end of all the upper insulating layers 113 can be relatively fixed to form a connection. The upper insulating layer 113 corresponding to the nucleic acid purification area 102 is a complete sheet-like insulating layer.

[0072] At least two different temperature zones are provided in the area corresponding to the nucleic acid amplification channel 1031, one being a high temperature zone and the other being a low temperature zone. The high temperature zone and the low temperature zone are within the range of the nucleic acid amplification channel 1031, so that during the reciprocating movement of the droplets in the nucleic acid amplification channel 1031, the droplets can reciprocate into different temperature zones and transfer in different temperature zones, thereby realizing rapid temperature change of the droplets.

[0073] The temperature range includes a high-temperature zone of no less than 60°C. The PCR process generally consists of 45 amplification cycles, each of which includes three stages: denaturation (approximately 95°C), annealing (approximately 60°C), and extension (approximately 72°C). The annealing and extension stages can be optimized to run at the same temperature, combining them into a single stage. Therefore, a high-temperature zone and a low-temperature zone can be set. The high-temperature zone has a higher temperature than the low-temperature zone, and the high-temperature and low-temperature zones do not indicate the absolute difference in temperature.

[0074] An optical signal collection area is provided in the area corresponding to the nucleic acid amplification flow channel 1031 for optical equipment to collect signals. An optical measurement is performed each time the droplet reciprocates and passes through the optical signal collection area, and a graph is drawn based on multiple measurement results.

[0075] The bubble capture structure 1032 is provided on both sides of the nucleic acid amplification flow channel 1031. Figure 3 、 Figure 14 As shown, two bubble capture structures 1032 are set on both sides of each nucleic acid amplification channel 1031. The bubble capture structure 1032 is located above the side of the nucleic acid amplification channel 1031. When the droplets move back and forth, the droplets push the bubbles to the vicinity of the capture structure 1032, and the bubbles will automatically be discharged upward into the bubble capture structure 1032, thereby eliminating the bubbles in the nucleic acid amplification channel 1031.

[0076] Each nucleic acid amplification channel 1031 is embedded with dried active materials, primers, and probes. The purpose of drying and embedding the active materials is to enable the cartridge to be stored at room temperature without significantly changing the activity of the reagents. After the PCR pre-reaction droplets and the dried active materials, primers, and probes are reconstituted, the PCR amplification reaction is initiated. The nucleic acid amplification channel 1031 is provided with reaction temperature zones of varying temperatures. By moving the droplets between these temperature zones, rapid temperature changes are achieved to complete the PCR amplification reaction. At the same time, bubble capture structures are provided on both sides of each nucleic acid amplification channel to capture bubbles generated during the PCR reaction; the waste liquid area is used to store waste liquid during the reagent reaction process.

[0077] In some embodiments, the thickness of the upper insulating layer 113 is 0.1 μm-20 μm, specifically 3 μm-15 μm, and further 5 μm-10 μm, which is suitable for droplet manipulation processes with a volume of 20 μL or more.

[0078] To facilitate engineering, the upper insulating layer 113 in the present invention utilizes a 9μm thick polyethylene terephthalate film tape, comprising a 3μm polyethylene terephthalate film and a 6μm adhesive backing. If the insulating layer is too thin, the droplets may not adhere to the upper plate or may have poor adhesion, resulting in a less three-dimensional droplet or an unstable shape. If the insulating layer is too thick, the effective electric field for driving the droplets is severely weakened, resulting in insufficient driving force and ultimately causing drive failure.

[0079] Combine Figure 1 、 Figure 2 As shown, the electrowetting microfluidic cartridge of the present invention further includes a reagent storage module 20, which can be used to store liquids such as reagents and samples. The reagent storage module 20 is located above the reagent dispensing area 101, and the liquid in the reagent storage module 20 can flow downward into the reagent dispensing area 101.

[0080] Combine Figure 7 As shown, the reagent storage module 20 of the present invention includes a skeleton 210 and a top cover 220 assembled with each other, and the skeleton 210 and the top cover 220 together constitute the main structure of the reagent storage module 20.

[0081] The skeleton 210 is provided with a plurality of storage cavities 211 , in which liquids such as reagents and samples can be placed. The storage cavity 211 is a vertically opened channel, and an aluminum-plastic film 212 is provided at the bottom of the storage cavity 211 . When the aluminum-plastic film 212 is broken, the liquid stored therein can be released.

[0082] The top cover 220 itself is a rigid structure, with flexible membranes 221 disposed in localized areas of the top cover 220. Each flexible membrane 221 corresponds to a storage cavity 211, and at least the upper ends of some storage cavities 211 are correspondingly provided with flexible membranes 221. The storage cavities 211 for storing samples are provided with sample plugs 213.

[0083] When the aluminum-plastic film 212 at the bottom of the storage cavity 211 ruptures, the air pressure inside the storage cavity 211 is lower than the external atmospheric pressure because its upper end is sealed by the flexible film 221. By pressing the flexible film 221, the internal volume of the storage cavity 211 can be reduced, causing the reagent in the corresponding storage cavity 211 to be discharged into the reagent dispensing area 101. In the reagent dispensing area 101, the driving electrode 124 is used to divide and drive the liquid to move.

[0084] Combine Figure 4 、 Figure 5 As shown, the upper substrate 111 is provided with a puncture column 1111, which protrudes upward from the upper substrate 111, and a sharp end is provided at the top of the puncture column 1111. The puncture column 1111 is used to puncture the aluminum-plastic film 212 provided at the bottom end of the storage cavity 211 when the reagent storage module 20 is pressed down.

[0085] Before testing, the reagent storage module 20 and the reagent reaction module 10 are assembled together, but the liquid reagent is sealed in the reagent storage module 20 and isolated from the reagent reaction module 10. The reagent storage module 20 is placed above the reagent reaction module 10. When testing is required, the reagent storage module 20 is pressed downward, causing the puncture posts 1111 to pierce the aluminum-plastic film 212 at the bottom of each storage cavity 211 and squeeze the flexible film 221 to release the liquid.

[0086] The top cover 220 is provided with an air hole 222, and the upper end of the air hole 222 is matched with an air hole plug 223. Before the reagent storage module 20 is pressed downward, the air hole plug 223 has not yet sealed the air hole 222. When the reagent storage module 20 is pressed downward and the aluminum-plastic film 212 is punctured, the air hole 222 is sealed, thereby achieving the sealing of the reagent storage module 20 and further achieving the full sealing of the entire card box.

[0087] Conventional electrowetting struggles to manipulate large droplets. It generates numerous bubbles at high temperatures, hindering actuation and optical signal acquisition. The present invention, by adding a thick insulating layer (i.e., upper insulating layer 113) to the upper plate 110, renders the droplets three-dimensional, enabling stable manipulation of larger reagent volumes. This prevents large droplets from being pulled during actuation, leading to severe tailing and resulting in liquid breakage. Driving stability is improved, enabling rapid and stable actuation of droplets of several hundred microliters. In this case, 650 μL of liquid can be stably actuated within a 2.5 mm high droplet channel. Conventional microfluidic chips typically have a height of less than 0.5 mm and actuation volumes of less than 20 μL. This also reduces the problem of bubbles caused by the superposition of electric and thermal fields during high-temperature reagent reactions. Furthermore, the more three-dimensional droplets facilitate accurate optical signal acquisition.

[0088] The present invention uses electrowetting technology to construct a fully integrated microfluidic cartridge, and designs different electrowetting structures in different reagent processing areas. For the nucleic acid purification area of ​​large-volume reaction reagents, an insulating layer is introduced into the traditional upper plate structure to change the electric field distribution, so that the droplets are transformed from a flat state to a three-dimensional state, thereby avoiding the droplets from being broken due to pulling during rapid driving, and achieving stable driving of large-volume reagents; for the nucleic acid amplification area, an insulating layer is also introduced into the upper plate to avoid the bubble problem caused by the Taylor cone effect generated by the electric field when the droplets are in a flat state. At the same time, the three-dimensional droplets are more likely to achieve accurate fluorescence signal acquisition; in the reagent distribution area, the traditional electrowetting upper plate structure design without an insulating layer is used, which can retain the effective electric field for droplet driving to the greatest extent, provide maximum driving force, and at the same time, when the sample and reagent are quantitatively distributed, the mother droplets can be smoothly broken and the daughter droplets of the preset volume can be accurately separated.

[0089] This invention achieves fully enclosed, integrated integration of sample preparation, nucleic acid purification, and PCR amplification detection, supporting rapid, highly sensitive detection of multiple nucleic acids. This electrowetting microfluidic cartridge (chip) structure is applicable not only to nucleic acid detection processes, but also to other scenarios requiring large-volume reagent manipulation or high-temperature reactions on an electrowetting chip.

[0090] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. An electrowetting microfluidic cartridge, characterized in that: The reagent reaction module (10) comprises an upper electrode plate (110) and a lower electrode plate (120) which are sealed and assembled with each other, and a liquid droplet channel is formed between the upper electrode plate (110) and the lower electrode plate (120); The upper electrode plate (110) comprises an upper substrate (111), an upper conductive layer (112), an upper insulating layer (113), and an upper hydrophobic layer (114); The upper insulating layer (113) is provided in at least a local area between the upper conductive layer (112) and the upper hydrophobic layer (114); the thickness of the upper insulating layer (113) is 0.1 μm-20 μm; The lower electrode plate (120) comprises a lower substrate (121), a lower insulating layer (122) and a lower hydrophobic layer (123); a driving electrode (124) is provided on the lower substrate (121).

2. The electrowetting microfluidic cartridge according to claim 1, wherein: The reagent reaction module (10) is provided with a reagent distribution area (101), a nucleic acid purification area (102), a nucleic acid amplification area (103) and a waste liquid area (104); The upper insulating layer (113) is arranged in the nucleic acid purification area (102) and / or the nucleic acid amplification area (103).

3. The electrowetting microfluidic cartridge according to claim 2, wherein: The nucleic acid amplification area (103) is provided with a plurality of nucleic acid amplification flow channels (1031) distributed side by side, and each nucleic acid amplification flow channel (1031) is used for flow of droplets; The upper insulating layer (113) is provided in a corresponding area of ​​each nucleic acid amplification flow channel (1031).

4. The electrowetting microfluidic cartridge according to claim 3, wherein: At least two different temperature zones are provided in the region corresponding to the nucleic acid amplification flow channel (1031), and the droplets move back and forth in the nucleic acid amplification flow channel (1031) to be transferred in different temperature zones.

5. The electrowetting microfluidic cartridge according to claim 4, wherein: The temperature region includes a high temperature zone not lower than 60°C.

6. The electrowetting microfluidic cartridge according to claim 3, wherein: An optical signal collection area is provided in the area corresponding to the nucleic acid amplification flow channel (1031) for optical equipment to collect signals.

7. The electrowetting microfluidic cartridge according to claim 3, wherein: Bubble capture structures (1032) are provided on both sides of the nucleic acid amplification flow channel (1031).

8. The electrowetting microfluidic cartridge according to any one of claims 2 to 7, wherein: It also includes a reagent storage module (20), wherein the reagent storage module (20) is located above the reagent distribution area (101); The reagent storage module (20) comprises a frame (210) and a top cover (220) assembled with each other, the frame (210) is provided with a plurality of storage cavities (211), and an aluminum-plastic film (212) is provided at the bottom end of the storage cavity (211); A flexible membrane (221) is provided on the top cover (220), and the flexible membrane (221) is correspondingly provided at the upper end of at least part of the storage cavity (211); a sample plug (213) is provided in the storage cavity (211) for storing samples.

9. The electrowetting microfluidic cartridge according to claim 8, wherein: The upper substrate (111) is provided with a puncture column (1111) for puncturing the aluminum-plastic film (212) provided at the bottom end of the storage cavity (211) when the reagent storage module (20) is pressed downward.

10. The electrowetting microfluidic cartridge according to claim 9, wherein: The top cover (220) is provided with an air hole (222), and an air hole plug (223) is provided at the upper end of the air hole (222); the air hole plug (223) is used to seal the air hole (222) after the reagent storage module (20) is pressed down and punctured.

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