An electrowetting microfluidic card

Abstract

The application discloses an electrowetting microfluidic card box, and relates to the technical field of electrowetting microfluidic control. An upper pole plate comprises an upper substrate, an upper conductive layer and an upper hydrophobic layer. An upper insulating layer is arranged in at least a partial region between the upper conductive layer and the upper hydrophobic layer. A lower pole plate comprises a lower substrate, a lower insulating layer and a lower hydrophobic layer. The lower substrate is provided with a driving electrode. A driving liquid moves in a liquid drop channel. In the region provided with the upper insulating layer, the upper part of the liquid drop contacts the upper hydrophobic layer and is insulated from the upper conductive layer, so that the electric charge of the upper conductive layer cannot be conducted to the liquid drop. Meanwhile, the lower part of the liquid drop contacts the lower hydrophobic layer and is insulated by the lower insulating layer, so that the liquid drop as a whole maintains a higher height, the liquid drop is more three-dimensional, the liquid drop is more easily driven, and bubbles are not easily generated during driving. In addition, the liquid drop is more closely in contact with the upper hydrophobic layer, the contact area of the liquid drop with the upper hydrophobic layer is larger, and optical detection of the liquid drop is more accurate.

Description

Technical Field

[0001] This invention relates to the field of electrowetting microfluidic control technology, and more particularly to an electrowetting microfluidic card. Background Technology

[0002] Closed microfluidic cartridges that integrate sample preparation, nucleic acid purification, and PCR (polymerase chain reaction) amplification and detection represent one of the main development directions in current molecular diagnostic technologies. With the continuous improvement of product performance requirements, achieving rapid and highly sensitive detection of ultra-multiplex nucleic acids in fully integrated microfluidic cartridges has become a crucial objective.

[0003] Currently, the main factor limiting detection speed is the temperature rise and fall rate during PCR amplification, while the key to achieving highly sensitive detection lies in the precise control of the reagent reaction process.

[0004] Electrowetting technology eliminates the need for precise temperature control of a specific area. Instead, it allows for the setting of different temperature zones, enabling droplets to move between these zones and thus achieving temperature increases or decreases. Due to its precise and flexible manipulation of tiny droplets, electrowetting technology can control the rapid switching of droplets between different PCR temperature zones, resulting in rapid temperature changes and significantly shortening PCR reaction time. Therefore, it has become 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 can cause serious bubble problems. The more bubbles there are, the more likely it is to cause droplet drive failure and inaccurate acquisition of reaction fluorescence signals.

[0006] Furthermore, to further improve the sensitivity and detection rate of nucleic acid testing, commercially available methods typically increase the sample volume during sample preparation and nucleic acid purification to obtain more target nucleic acids for PCR amplification and detection. However, the advantage of traditional electrowetting systems lies in the control of small droplets, making it difficult to quickly and stably manipulate large droplets of 20 μL or more, and even more so, it cannot be compatible with sample preparation and nucleic acid purification processes of several hundred microliters.

[0007] Therefore, traditional electrowetting PCR produces serious bubble problems, affecting driving stability and the accuracy of fluorescence signal acquisition; moreover, traditional electrowetting cannot control large droplets. Summary of the Invention

[0008] The core of this invention is to provide an electrowetting microfluidic cartridge. By providing an upper insulating layer in at least a localized area between the upper conductive layer and the upper hydrophobic layer, the droplet remains electrically neutral in the area covered by the upper insulating layer, maintaining a more three-dimensional shape. This enables control of large droplets, reduces bubble formation, and improves photometric accuracy. 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 together, 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 disposed in at least a localized area 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 a driving electrode is provided on the lower substrate.

[0013] Optionally, the reagent reaction module includes a reagent dispensing area, a nucleic acid purification area, a nucleic acid amplification area, and a waste liquid area;

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

[0015] Optionally, the nucleic acid amplification region is provided with multiple nucleic acid amplification channels arranged side by side, each of the nucleic acid amplification channels being used for droplet flow;

[0016] Each of the nucleic acid amplification channels has an upper insulating layer in its corresponding region.

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

[0018] Optionally, the temperature range includes a high-temperature zone of not less than 60°C.

[0019] Optionally, the area corresponding to the nucleic acid amplification channel is provided with an optical signal acquisition area for optical equipment to acquire signals.

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

[0021] Optionally, it also includes a reagent storage module located above the reagent dispensing area;

[0022] The reagent storage module includes a frame and a top cover that are assembled together. The frame is provided with multiple storage cavities, and an aluminum-plastic film is provided at the bottom of each storage cavity.

[0023] A flexible membrane is provided on the top cover, and the flexible membrane is provided at least partially on the upper end of the storage cavity; a sample plug is provided in the storage cavity for storing the sample.

[0024] Optionally, the upper substrate is provided with a piercing post for piercing the aluminum-plastic film at the bottom of the storage cavity when the reagent storage module is pressed down.

[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] This invention provides an electrowetting microfluidic cartridge, wherein the reagent reaction module includes an upper electrode plate and a lower electrode plate that are sealed together, forming a droplet channel for droplet flow. The upper electrode plate includes an upper substrate, an upper conductive layer, and an upper hydrophobic layer, with an upper insulating layer disposed in at least a localized area between the upper conductive layer and the upper hydrophobic layer. The lower electrode plate includes a lower substrate, a lower insulating layer, and a lower hydrophobic layer, with a driving electrode disposed on the lower substrate to drive the liquid to move in the droplet channel. Because an upper insulating layer is disposed in a localized 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 disposed. The hydrophobic layer forms an insulation with the upper conductive layer, preventing the charge of the upper conductive layer from being conducted to the droplet. Simultaneously, the lower part of the droplet contacts the lower hydrophobic layer and forms an insulation through the lower insulating layer. The droplet remains electrically neutral in the region with the upper insulating layer and forms a three-dimensional shape under the influence of surface tension and electric field, thus maintaining a higher overall height of the droplet. The droplet is easier to drive and less prone to breakage. Due to the higher three-dimensionality of the droplet, it is less likely to generate bubbles during driving. Furthermore, because the droplet has a closer contact with the upper hydrophobic layer and a larger contact area, optical detection of the droplet is more accurate. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 This is a schematic diagram of the overall structure of the electrowetting microfluidic cartridge of the present invention;

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

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

[0031] Figure 4 This is a diagram showing the relative relationship between the upper and lower electrodes;

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

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

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

[0035] Figure 8A This is a front view of the droplet morphology without an insulating layer on the upper electrode plate.

[0036] Figure 8B A top view of the upper electrode plate without an insulating layer, driven by a large droplet to break it;

[0037] Figure 9A A front view of the droplet morphology with an insulating layer on the upper electrode;

[0038] Figure 9B A top-view schematic diagram showing a large-volume droplet driving a normal operation with an insulating layer on the upper electrode.

[0039] Figure 10A A frontal view of the droplet-driven morphology and bubble generation of the upper electrode plate without an insulating layer;

[0040] Figure 10B Top view of the droplet-driven morphology and bubble generation of the upper electrode plate without an insulating layer;

[0041] Figure 11A A frontal view of the upper electrode plate with an insulating layer, showing the droplet-driven morphology and bubble generation.

[0042] Figure 11B Top view showing the droplet-driven morphology and bubble generation of the upper electrode plate with an insulating layer;

[0043] Figure 12A A frontal view of the upper electrode plate without an insulating layer, where the droplet is blocked by a bubble.

[0044] Figure 12B Top view of the upper electrode without an insulating layer - droplets blocked by air bubbles;

[0045] Figure 13 A schematic diagram illustrating the optical measurements of four different droplet shapes;

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

[0047] The image includes:

[0048] The reagent reaction module 10, reagent dispensing area 101, nucleic acid purification area 102, nucleic acid amplification area 103, nucleic acid amplification channel 1031, bubble trapping structure 1032, and waste liquid area 104 are included.

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

[0050] Reagent storage module 20, skeleton 210, storage cavity 211, aluminum-plastic film 212, sample plug 213, top cover 220, flexible film 221, pores 222, pore plug 223. Detailed Implementation

[0051] 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 embodiments.

[0052] This 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 the driving control of the liquid.

[0053] Electrowetting microfluidics eliminates the need for pumps, valves, and tubing. By applying an external electric field, it alters the contact angle between the solid and liquid phases, causing asymmetric deformation of the droplets and thus enabling precise droplet manipulation. Because the input voltage of the driving electrode array can be flexibly controlled via digital programming, it is also known as digital microfluidics.

[0054] Combination Figure 4 As shown, the reagent reaction module 10 includes an upper electrode plate 110 and a lower electrode plate 120 that are sealed together. A sealing ring 130 is provided between the upper electrode plate 110 and the lower electrode plate 120 to keep the gap between them sealed for circumference. A droplet channel is formed between the upper electrode plate 110 and the lower electrode plate 120, allowing the droplet to move within the gap. Specifically, the upper hydrophobic layer 114 of the upper electrode plate 110 contacts the droplet, and the lower hydrophobic layer 123 of the lower electrode plate 120 contacts the droplet.

[0055] Combination Figure 5As shown, the upper electrode 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, upper conductive layer 112, upper insulating layer 113, and upper hydrophobic layer 114 are arranged in a stacked relationship, from top to bottom as upper substrate 111, upper conductive layer 112, upper insulating layer 113, and upper hydrophobic layer 114. The upper insulating layer 113 serves 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 partial 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; it only needs to be disposed in a portion of the area. Of course, if necessary, the present invention allows the upper insulating layer 113 to be disposed in all of the aforementioned areas.

[0057] Combination Figure 6 As shown, in order from bottom to top, the lower electrode 120 includes 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, and the driving electrode 124 is formed by a plurality of electrode blocks arranged in a predetermined pattern on the lower substrate 121. 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 areas of the lower insulating layer 122 and the lower substrate 121 are approximately equal. The lower insulating layer 122 forms an insulation layer for every area of ​​the lower electrode 120, ensuring that the droplet does not conduct electricity with the driving electrode 124 regardless of its position.

[0058] In this invention, an upper insulating layer 113 is provided in a local area of ​​the upper electrode plate 110, which insulates the upper conductive layer 112. A 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 110 does not have an upper insulating layer 113, or in areas 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 become charged. This charge has the opposite polarity to the charge of the driving electrode 124. Since opposite charges attract each other, the driving electrode 124 attracts the droplet, causing it to move closer to the lower hydrophobic layer 123, resulting in a droplet with a smaller top and a larger bottom shape (e.g., ...). Figure 8A As shown, the area of ​​the droplet contacting the lower hydrophobic layer 123 is much larger than the area contacting the upper hydrophobic layer 114. Because of the large contact area between the droplet and the lower hydrophobic layer 123, and the attraction from the driving electrode 124, the droplet is prone to breakage when driven by the driving electrode 124, making it difficult to maintain the shape of a single droplet. Figure 8BAs shown, the motion of a 650 μL large-volume droplet is illustrated. An image is acquired every 0.6 s, with images taken at time points of 0.6 s, 1.2 s, 1.8 s, 2.4 s, 3.0 s, and 3.6 s during the droplet's motion, demonstrating the droplet breakage process. Without the upper insulating layer 113, only small-volume droplets can be driven. Large-volume droplets, due to their large size and lower three-dimensionality, spread out on the lower hydrophobic layer 123. The contact area between the droplet and the lower hydrophobic layer 123 is larger, making it more difficult to synchronously drive the droplet to move at different positions. Maintaining the shape of a single droplet during motion is also more challenging, making droplet breakage more likely.

[0060] In this invention, an upper insulating layer 113 is disposed between the upper conductive layer 112 and the upper hydrophobic layer 114. The upper insulating layer 113 forms an electrical insulation between the upper conductive layer 112 and the upper conductive layer 113. The upper end of the droplet is no longer charged by the upper conductive layer 112, while the lower end of the droplet is insulated from the driving electrode 124 through the lower insulating layer 122. The droplet is almost unaffected by the charges of the upper conductive layer 112 and the driving electrode 124, maintaining its electrical neutrality. Under electrical neutrality, the droplet, under the influence of its surface tension and electric field, exhibits a full three-dimensional shape (e.g., ...). Figure 9A As shown in the diagram, the contact states between the droplet and the upper hydrophobic layer 114 and between the droplet and the lower hydrophobic layer 123 are almost identical. The area of ​​the droplet contacting the upper hydrophobic layer 114 is approximately equal to the area of ​​the droplet contacting the lower hydrophobic layer 123. The smaller area of ​​the droplet contacting the lower hydrophobic layer 123 makes it less likely for the droplet to break when driven to move. Therefore, large-volume droplets can be driven in the area where the upper insulating layer 113 is located. Figure 9B As shown, the motion of a 650 μL large-volume droplet was demonstrated. An image was acquired every 0.6 s. Images were taken at time points of 0.6 s, 1.2 s, 1.8 s, 2.4 s, 3.0 s, and 3.6 s during the droplet's motion. Throughout the entire motion of the droplet, there was no breakage of the droplet, and it remained a complete droplet.

[0061] Combination Figure 10A and Figure 10B As shown, the droplet driving pattern and bubble generation are illustrated when the upper electrode 110 has no upper insulating layer 113. When the droplet is in a flat shape, it adheres more closely to the lower hydrophobic layer 123. The Taylor cone effect at the top of the droplet caused by the electric field leads to bubble formation.

[0062] Combination Figure 11A and Figure 11B As shown, the droplet driving pattern and bubble generation are illustrated when the upper insulating layer 113 is provided on the upper electrode 110. When the droplet is in a three-dimensional shape, the contact area between the upper end of the droplet and the upper hydrophobic layer 114 is larger, which reduces the Taylor cone effect at the upper end of the droplet caused by the electric field, thereby significantly reducing the number of bubbles generated during the PCR process.

[0063] Combination Figure 12A and Figure 12B As shown, Figure 12A Images (A), (B), and (C) sequentially illustrate the process of droplets being blocked by air bubbles; flat droplets are more prone to incorporating gas during movement, making them more likely to break apart (e.g., Figure 12B (As shown). In this invention, an insulating layer 113 is provided on the upper electrode plate 110. For the small number of bubbles generated, the three-dimensional droplets can more easily push the small number of bubbles into the bubble capturing structures on both sides (see below for details), avoiding the droplets from being blocked by bubbles and failing to drive or being broken by bubbles.

[0064] Simultaneously, the three-dimensional droplet shape offers higher stability for fluorescence signal acquisition compared to the traditional electrowetting flattened shape. Combined with... Figure 13 As shown, the diagram illustrates the optical measurement states of four different droplet shapes: A, B, C, and D. Shapes A, B, and C represent droplets without the upper insulating layer 113 on the upper electrode 110. Because the upper end of the droplet cannot adhere tightly to the upper hydrophobic layer 114 to form gaps or air bubbles, the detection light cannot easily penetrate the droplet, making high-precision detection difficult. Shape D represents a droplet with the upper insulating layer 113 on the upper electrode 110. Because the droplet is more three-dimensional, its upper end can adhere tightly to the upper hydrophobic layer 114, allowing the detection light to better illuminate the droplet's interior, facilitating accurate optical signal acquisition.

[0065] Therefore, it can be seen that by setting an upper insulating layer 113 between the upper conductive layer 112 and the upper hydrophobic layer 114, the present invention can produce three major technical effects: 1) it can drive larger volume droplets; 2) it reduces the generation of bubbles in droplets; and 3) it makes optical detection more accurate.

[0066] like Figure 3 As shown, this is equivalent to a top-down view; the entire reagent reaction module 10 is divided into a reagent dispensing area 101, a nucleic acid purification area 102, a nucleic acid amplification area 103, and a waste liquid area 104, with different areas divided according to function. Figure 3 The view shown from above depicts different areas, which are essentially divided into different parts on a horizontal plane, and these different areas are adjacent to each other.

[0067] The reagent dispensing area 101 is used to manipulate the sample and liquid reagent to complete the precise quantitative dispensing, and then enters the nucleic acid purification area 102. The nucleic acid purification area 102 is pre-embedded with dried magnetic beads, which work with the liquid reagent to complete the nucleic acid purification process. After the purified nucleic acid is mixed with PCR buffer, it is further quantitatively separated into several PCR pre-reaction droplets through the reagent dispensing area 101 and enters the nucleic acid amplification area 103.

[0068] The upper substrate 111, the upper conductive layer 112, and the upper hydrophobic layer 114 are disposed over the entire area of ​​the upper electrode plate 110, in combination with... Figure 5 As shown, the upper substrate 111, the upper conductive layer 112, and the upper hydrophobic layer 114 have approximately equal projected areas on the horizontal plane, respectively covering the reagent dispensing area 101, the nucleic acid purification area 102, the nucleic acid amplification area 103, and the waste liquid area 104. The sealing area does not have a hydrophobic layer (as a hydrophobic layer would affect the sealing), and other non-droplet manipulation areas, such as the waste liquid area 104, the bubble trapping structure 1032, and the sealing area, may not have a conductive layer or a hydrophobic layer.

[0069] Based on the above scheme, combined with Figure 3 , Figure 5 As shown, the upper insulating layer 113 is disposed in the nucleic acid purification area 102 and the nucleic acid amplification area 103, but not in the reagent dispensing area 101 and the waste liquid area 104. The upper insulating layer 113 helps to improve the three-dimensionality of the droplets, which can drive larger droplets and makes it less likely for the droplets to break apart. Since the reagent dispensing area 101 needs to quantitatively extract a certain volume of droplets, the upper insulating layer 113 is not required in the reagent dispensing area 101 to ensure that the reagent dispensing area 101 can accurately extract droplets.

[0070] The sample is purified in the nucleic acid purification zone 102 by magnetic beads to adsorb nucleic acid, thus achieving nucleic acid aggregation. Other liquids are discharged into the waste liquid zone 104. The waste liquid zone 104 has no requirements on the shape of the droplets, so there is no need to install an insulating layer 113.

[0071] The nucleic acid amplification region 103 has multiple nucleic acid amplification channels 1031 arranged side by side, each of which is used to supply droplet flow; combined with Figure 3 As shown, a total of 8 nucleic acid amplification channels 1031 are provided, each channel 1031 allowing one droplet to reciprocate, with the direction of movement as indicated by the bidirectional arrows. Each nucleic acid amplification channel 1031 has an upper insulating layer 113 in its corresponding region, as shown... Figure 5 As shown, each nucleic acid amplification channel 1031 is provided with an upper insulating layer 113. For ease of assembly, one end of all the upper insulating layers 113 can be fixed relative to each other for connection. The upper insulating layer 113 corresponding to the nucleic acid purification zone 102 is a complete sheet-like insulating layer.

[0072] At least two different temperature zones are provided in the region corresponding to the nucleic acid amplification channel 1031, one is a high temperature zone and the other is a low temperature zone. The high temperature zone and the low temperature zone are located within the range of the nucleic acid amplification channel 1031, so that the droplet can repeatedly enter different temperature zones and transfer in different temperature zones during the reciprocating movement in the nucleic acid amplification channel 1031, thereby achieving rapid temperature change of the droplet.

[0073] The temperature range includes a high-temperature zone of at least 60°C. A typical PCR process consists of 45 amplification cycles, each including three phases: denaturation (approximately 95°C), annealing (approximately 60°C), and extension (approximately 72°C). The annealing and extension phases can be optimized to be performed at the same temperature, thus merging into a single phase. Therefore, a high-temperature zone and a low-temperature zone can be defined. The high-temperature zone has a higher temperature than the low-temperature zone; however, the terms "high-temperature zone" and "low-temperature zone" do not represent absolute temperature differences.

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

[0075] The nucleic acid amplification channel 1031 has bubble trapping structures 1032 on both sides, which bind to... Figure 3 , Figure 14 As shown, two bubble trapping structures 1032 are provided on both sides of each nucleic acid amplification channel 1031. The bubble trapping structures 1032 are located above the side of the nucleic acid amplification channel 1031. When the droplet moves back and forth, the droplet pushes the bubble to the vicinity of the trapping structure 1032. The bubble will automatically be discharged upward into the bubble trapping structure 1032, thereby eliminating the bubble in the nucleic acid amplification channel 1031.

[0076] Each nucleic acid amplification channel 1031 is embedded with dried bioactive material, primers, and probes. The drying and embedding of the bioactive material ensures the cartridges can be stored at room temperature without significant changes in reagent activity. After the PCR pre-reaction droplets are reconstituted with the dried bioactive material, primers, and probes, the PCR amplification reaction is initiated. The nucleic acid amplification channel 1031 has different temperature zones; rapid temperature changes are achieved through droplet movement within these zones to complete the PCR amplification reaction. Simultaneously, each nucleic acid amplification channel has bubble-capturing structures on both sides to capture bubbles generated during the PCR reaction; a waste liquid area is used for storing waste liquid generated during the reagent reaction process.

[0077] In some embodiments, the thickness of the upper insulating layer 113 is 0.1 μm-20 μm, specifically between 3 μm-15 μm, and further between 5 μm-10 μm. This is suitable for droplet manipulation processes with volumes of 20 μL and above.

[0078] From an engineering perspective, the upper insulating layer 113 in this invention uses a polyethylene terephthalate (PET) film tape with a total thickness of 9 μm, comprising a 3 μm PET film and a 6 μm adhesive backing. If the insulating layer thickness is too small, the droplet may not adhere tightly to the upper electrode or may have poor adhesion, resulting in a less three-dimensional droplet or an unstable shape. Conversely, if the insulating layer thickness is too large, the effective electric field driving the droplet will be severely weakened, leading to insufficient driving force and ultimately causing driving failure.

[0079] Combination Figure 1 , Figure 2 As shown, the electrowetting microfluidic cartridge of the present invention also 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] Combination Figure 7 As shown, the reagent storage module 20 of the present invention includes a frame 210 and a top cover 220 assembled together, and the frame 210 and the top cover 220 together constitute the main structure of the reagent storage module 20.

[0081] The skeleton 210 is provided with multiple storage cavities 211, which can hold reagents, samples and other liquids. The storage cavities 211 are vertically opened channels, and the bottom of the storage cavities 211 is provided with an aluminum-plastic film 212. 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, and a flexible membrane 221 is provided in a local area on the top cover 220. Each flexible membrane 221 corresponds to a storage cavity 211, and the upper end of at least part of the storage cavity 211 is provided with a flexible membrane 221. The storage cavity 211 used to store samples is provided with a sample plug 213.

[0083] When the aluminum-plastic film 212 at the bottom of the storage cavity 211 breaks, 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. The internal volume of the storage cavity 211 can be reduced by pressing the flexible film 221, so that the reagent in the corresponding storage cavity 211 is discharged into the reagent distribution area 101. In the reagent distribution area 101, the liquid is divided and driven to move by the driving electrode 124.

[0084] Combination Figure 4 , Figure 5 As shown, the upper substrate 111 is provided with a piercing post 1111, which protrudes upward from the upper substrate 111. The top of the piercing post 1111 is provided with a sharp end. The piercing post 1111 is used to pierce the aluminum-plastic film 212 provided at the bottom 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 down, causing the piercing column 1111 to pierce the aluminum-plastic film 212 at the bottom of each storage cavity 211 and squeeze the flexible membrane 221 to release the liquid.

[0086] 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. Before the reagent storage module 20 is pressed down, the air hole plug 223 has not yet sealed the air hole 222. When the reagent storage module 20 is pressed down and punctures the aluminum-plastic film 212, the air hole 222 is sealed, thereby achieving the airtightness of the reagent storage module 20 and thus achieving the complete sealing of the entire card box.

[0087] Traditional electrowetting struggles to control large droplets, and at high temperatures, it generates numerous bubbles, hindering actuation and optical signal acquisition. This invention adds a layer of insulation (upper insulating layer 113) of a certain thickness to the upper electrode 110, creating a three-dimensional droplet. This allows for stable control of larger reagent volumes, preventing the large droplets from being stretched or severely tailed during actuation, thus avoiding liquid interruption. Actuation stability is improved, enabling rapid and stable actuation of droplets up to several hundred microliters. In this case, stable actuation of 650 μL of liquid can be achieved within a droplet channel only 2.5 mm high, while traditional microfluidic chips typically have a height of less than 0.5 mm and an actuation volume of less than 20 μL. Simultaneously, it reduces the bubble problem caused by the superposition of electric and thermal fields during high-temperature reagent reactions; the more three-dimensional droplet also facilitates precise optical signal acquisition.

[0088] This invention employs electrowetting technology to construct a fully integrated microfluidic cartridge, designing different electrowetting structures in different reagent processing areas. For the nucleic acid purification area with large-volume reaction reagents, an insulating layer is introduced into the traditional upper electrode structure to alter the electric field distribution, transforming the droplet from a flat state to a three-dimensional state. This prevents the droplet from breaking due to stretching during rapid actuation, achieving stable actuation of large-volume reagents. Similarly, for the nucleic acid amplification area, an insulating layer is introduced into the upper electrode to prevent bubble formation caused by the Taylor cone effect of the electric field when the droplet is flat. Simultaneously, the three-dimensional droplet facilitates more accurate fluorescence signal acquisition. In the reagent dispensing area, the traditional electrowetting upper electrode without an insulating layer is used, maximizing the preservation of the effective electric field for droplet actuation and providing maximum driving force. Furthermore, during quantitative sample and reagent dispensing, the mother droplet breaks smoothly, accurately separating sub-droplets of a predetermined volume.

[0089] This invention achieves a fully enclosed, integrated process for sample preparation, nucleic acid purification, and PCR amplification and detection, supporting rapid and highly sensitive detection of multiplex nucleic acids. This electrowetting microfluidic cartridge (chip) structure can be applied 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 enables those skilled in the art to make or use the 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 invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded 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) includes an upper electrode plate (110) and a lower electrode plate (120) that are sealed together, and a droplet channel is formed between the upper electrode plate (110) and the lower electrode plate (120). 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 insulating layer (113) is disposed in a local area between the upper conductive layer (112) and the upper hydrophobic layer (114); the thickness of the upper insulating layer (113) is 0.1μm-20μm; The lower electrode plate (120) includes a lower substrate (121), a lower insulating layer (122) and a lower hydrophobic layer (123), and a driving electrode (124) is provided on the lower substrate (121). The reagent reaction module (10) is provided with a reagent dispensing 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 disposed in the nucleic acid purification region (102) and / or the nucleic acid amplification region (103).

2. The electrowetting microfluidic cartridge according to claim 1, characterized in that, The nucleic acid amplification region (103) is provided with multiple nucleic acid amplification channels (1031) arranged side by side, and each of the nucleic acid amplification channels (1031) is used for droplet flow; Each of the nucleic acid amplification channels (1031) has an upper insulating layer (113) in its corresponding region.

3. The electrowetting microfluidic cartridge according to claim 2, characterized in that, At least two different temperature zones are provided in the region corresponding to the nucleic acid amplification channel (1031), and the droplet moves back and forth in the nucleic acid amplification channel (1031) to transfer between different temperature zones.

4. The electrowetting microfluidic cartridge according to claim 3, characterized in that, The temperature range includes a high-temperature zone of not less than 60°C.

5. The electrowetting microfluidic cartridge according to claim 2, characterized in that, The area corresponding to the nucleic acid amplification channel (1031) is provided with an optical signal acquisition area for optical equipment to acquire signals.

6. The electrowetting microfluidic cartridge according to claim 2, characterized in that, The nucleic acid amplification channel (1031) is provided with bubble trapping structures (1032) on both sides.

7. The electrowetting microfluidic cartridge according to any one of claims 1 to 6, characterized in that, It also includes a reagent storage module (20) located above the reagent dispensing area (101); The reagent storage module (20) includes a frame (210) and a top cover (220) assembled together. The frame (210) is provided with multiple storage cavities (211), and an aluminum-plastic film (212) is provided at the bottom of the storage cavity (211). A flexible membrane (221) is provided on the top cover (220), and the flexible membrane (221) is provided on 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.

8. The electrowetting microfluidic cartridge according to claim 7, characterized in that, The upper substrate (111) is provided with a piercing post (1111) for piercing the aluminum-plastic film (212) at the bottom of the storage cavity (211) when the reagent storage module (20) is pressed down.

9. The electrowetting microfluidic cartridge according to claim 8, characterized in that, 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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