Multi-sample parallel two-step PCR (Polymerase Chain Reaction) digital micro-fluidic chip and electrode layout

Abstract

The invention discloses a multi-sample parallel two-step method PCR digital micro-fluidic chip and electrode layout, the chip comprises a PCB substrate, a driving electrode, a dielectric water delivery layer and an upper cover plate which are sequentially distributed from bottom to top, the PCB substrate and the upper cover plate are sealed through glue, the dielectric water delivery layer is filled with sealing oil, and the driving electrode is arranged on the upper cover plate. Liquid drops which are not fused are wrapped in the sealing oil, and the electrode layout comprises a loading area, a mixed liquid separation area, a reaction area, a liquid injection and collection area, an oil seal sealing area and a conductive area; the chip adopts a PCB material as the basis of the micro-fluidic chip, so that not only is the production cost greatly reduced, but also the large-scale manufacturing and application of the chip are easier to realize, the electrode layout ensures that the micro-droplets can accurately and efficiently move and operate in the chip by optimizing the electrode arrangement, the design and control complexity is remarkably reduced, and the production efficiency is improved. The kit is designed to meet the requirements of two-step PCR, so that not only can high-efficiency transmission, mixing and separation of samples be realized, but also the reaction precision and efficiency can be ensured.

Description

Technical Field

[0001] The present invention belongs to the field of microfluidic chips, and more particularly, relates to a multi-sample parallel two-step PCR digital microfluidic chip and electrode layout. Background Art

[0002] Digital Microfluidics (DMF) is a microfluidic technology that precisely manipulates tiny droplets through the Electrowetting on Dielectric (EWOD) effect. Different from traditional continuous-flow microfluidics, digital microfluidic technology can control operations such as droplet generation, splitting, merging, and transportation on the open two-dimensional chip surface by applying an electric field. Due to its high flexibility and precise droplet control ability, digital microfluidics is widely used in biomedical analysis, drug screening, gene detection and other fields.

[0003] Polymerase Chain Reaction (PCR) is a technique for in vitro amplification of specific DNA sequences and has become a key tool in molecular biology and medical diagnosis. Traditional PCR includes three main steps: denaturation, annealing, and extension. When rapidly amplifying or simplifying the operation process, two-step PCR can be used. Two-step PCR combines the annealing and extension steps into one temperature step, only performing denaturation and extension. This method reduces the number of temperature conversions, shortens the reaction time, and improves the amplification efficiency. This two-step PCR shows higher specificity and sensitivity in multiplex detection, low-abundance sample analysis, etc.

[0004] Two-step PCR amplification on a digital microfluidic platform will fully utilize the advantages of both to achieve efficient nucleic acid amplification. First, through digital microfluidic technology, samples and PCR reaction mixtures are precisely dispensed into multiple independent droplets on the chip, and each droplet is processed as an independent reaction unit. In the first stage, the DNA sample in the droplet will be denatured at a high-temperature region, causing the double-stranded DNA to unwind into single strands to prepare for the amplification reaction. Subsequently, in the second stage, the droplet moves to the low-temperature region, where both annealing (primer binding to the target DNA) and extension (DNA polymerase synthesizing new DNA strands) occur. The advantage of the digital microfluidic system is that it can flexibly control multiple droplets to react simultaneously through an electrode array, realizing multi-sample parallel processing, and each droplet is independent of each other, greatly reducing the risk of cross-contamination. The whole process can be completed fully automatically, greatly improving the experimental efficiency and operation simplicity.

[0005] In the selection of materials for conventional digital microfluidic chips, several materials such as silicon and glass are mainly used. Due to its excellent thermal conductivity, chemical stability, and mechanical strength, silicon materials are commonly used in applications that require high-precision microfluidic structures and temperature control systems. However, the cost of silicon materials is relatively high, the manufacturing process is complex, and it is not suitable for low-cost mass production. Glass has excellent optical transparency and chemical stability, but its processing difficulty and cost are also relatively high. The present invention proposes a novel multi-sample parallel two-step PCR digital microfluidic chip.

[0006] There are many deficiencies in the design of the electrode layout in the prior art. First of all, the electrode distribution is usually not uniform enough to cover the entire droplet movement area, resulting in limited movement of the droplets and making it difficult to achieve efficient sample transfer and mixing. In addition, the electrode drive circuits of the existing layouts often require complex control logics, with relatively high operation difficulty and prone to misoperation, affecting the stability of experimental results. More critically, most of the existing layouts are designed for a single process and lack adaptability, making it difficult to meet the complex requirements of multi-stage temperature control and reagent dispensing in two-step PCR. Summary of the Invention

[0007] To address the above deficiencies, the present invention provides a multi-sample parallel two-step PCR digital microfluidic chip, which includes a PCB substrate, driving electrodes, a dielectric water conveyance layer, and an upper cover plate that are sequentially distributed from bottom to top. The PCB substrate and the upper cover plate are sealed with glue, and the dielectric water conveyance layer is filled with sealing oil, and immiscible droplets are encapsulated in the sealing oil;

[0008] The PCB substrate is configured with electrode lines for realizing the electro-wetting effect;

[0009] The driving electrodes are arranged on the PCB substrate and are used to control the loading, transfer, mixing, and liquid separation operations of the sample liquid;

[0010] The dielectric water conveyance layer includes a top layer and a bottom layer, and the top layer and the bottom layer are bonded and fixed to encapsulate and seal the sealing oil;

[0011] Both the sealing oil and the droplets are injected or extracted through the sample addition and sampling holes opened on the upper cover plate and the top layer.

[0012] Further, the driving electrode has a liquid carrying volume of 3 μL for a single electrode.

[0013] Further, the sealing oil is a highly stable hydrophobic insulating oil.

[0014] The present invention also discloses an electrode layout of a multi-sample parallel two-step PCR digital microfluidic chip, which includes the above-mentioned multi-sample parallel two-step PCR digital microfluidic chip, and further includes the following partitions:

[0015] Sample loading area for initial sample introduction;

[0016] Mixing and dispensing area for precise dispensing and mixing of liquids;

[0017] Reaction area with an independent temperature control unit for temperature adjustment;

[0018] Liquid injection and collection area for post-injection storage of liquids and collection and processing of final inspection liquids;

[0019] Oil seal and enclosure area for encapsulating sealing oil and preventing liquid from contacting the outside;

[0020] Conductive area for achieving electrode conduction and realizing the electro-wetting effect.

[0021] Further, the sample loading area is divided into a cold loading area and a hot loading area. The liquid injection and collection area includes cold loading holes and hot loading holes corresponding to the cold loading area and the hot loading area, and also includes sample holes for collection and processing of final inspection liquids.

[0022] Further, the triangular electrode structure in the mixing and dispensing area is controlled by logic electrodes to achieve right-angle dispensing to ensure the accuracy and uniformity of the dispensing process.

[0023] Further, the reaction area includes a high-temperature area and a low-temperature area that meet the requirements of the two-step PCR reaction, and controls the switching of droplets between the high- and low-temperature areas through electro-wetting path planning.

[0024] The present invention has the following beneficial effects compared with the prior art:

[0025] 1. Using PCB material as the basis of the microfluidic chip not only greatly reduces the production cost, but also makes it easier to achieve large-scale manufacturing and application of the chip;

[0026] 2. By optimizing the electrode arrangement layout, it is ensured that micro-droplets can move and operate precisely and efficiently within the chip, significantly reducing the design and control complexity. The design for the two-step PCR requirement can not only achieve efficient transmission, mixing and separation of samples, but also ensure the accuracy and efficiency of the reaction;

[0027] 3. The electrode layout of the present invention particularly supports parallel processing of multiple samples, can carry out multiple independent PCR experiments simultaneously on a single chip, thereby significantly improving the experimental throughput and working efficiency. At the same time, the reasonable electrode area division and optimized driving algorithm effectively reduce the energy consumption of the chip and improve the operation stability and applicability of the system;

[0028] 4. By optimizing the chip structure and electrode layout design, and combining the electro-wetting effect and path planning, the operation of multi-sample parallel two-step PCR reaction has been successfully achieved, which has the characteristics of high efficiency, precision, modularity, etc., and has broad application potential in medical testing, scientific research experiments, and portable molecular diagnostic devices. Description of the Drawings

[0029] Figure 1 It is a longitudinal structure schematic diagram of a multi-sample parallel two-step PCR digital microfluidic chip.

[0030] Figure 2 It is a schematic diagram of a multi-sample parallel two-step PCR digital microfluidic chip in the electrode conduction state.

[0031] Figure 3 It is a schematic diagram of the electrode layout of a multi-sample parallel two-step PCR digital microfluidic chip.

[0032] Figure 4 For Figure 3 detailed schematic diagram.

[0033] Figure 5 It is a PCB design diagram of a multi-sample parallel two-step PCR digital microfluidic chip.

[0034] In the figure: 1. PCB substrate; 2. Driving electrode; 3. Bottom layer; 4. Top layer; 5. Upper cover plate; 6. Sealing oil; 7. Droplet; 8. Glue; 9. Sampling and adding hole; 11. Sample loading area; 111. Cold loading area; 112. Hot loading area; 22. Mixing and liquid separation area; 33. Reaction area; 331. High temperature area; 332. Low temperature area; 44. Liquid injection and collection area; 441. Cold loading hole; 442. Hot loading hole; 443. Sample hole; 55. Oil seal closed area; 66. Conductive area. Detailed Embodiments

[0035] For the convenience of understanding the present invention, the device of the present invention will be described more comprehensively below with reference to the relevant drawings. Embodiments of the device are shown in the drawings. However, the device can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the disclosure of the present invention more thorough and comprehensive.

[0036] In the description of the present invention, it should be noted that unless otherwise clearly specified and limited, the terms "installation", "connection", and "setting" should be understood in a broad sense. For example, it can be fixedly connected and set, or detachably connected and set, or integrally connected and set. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific situations.

[0037] Embodiment 1

[0038] As Figure 1-2 shown, this embodiment provides a multi - sample parallel two - step PCR digital microfluidic chip, which includes a PCB substrate 1, driving electrodes 2, a dielectric water - transporting layer, and an upper cover plate 5 distributed in sequence from bottom to top. The PCB substrate 1 and the upper cover plate 5 are sealed by glue 8, and the dielectric water - transporting layer is filled with a sealing oil 6 (silicone oil is used). The sealing oil 6 encapsulates immiscible droplets 7. It should be noted that in addition to the stacked structure in this embodiment, the driving electrodes 2 and the control circuit can also be integrated into a single - layer structure, and the chip structure can be further simplified through specific processes (such as printed electronics technology). At the same time, the anti - pollution ability and reaction stability of the chip can be improved by adding nanomaterials into the dielectric water - transporting layer;

[0039] Specifically, the PCB substrate 1 is configured with electrode lines for realizing the electro - wetting effect to ensure the efficient operation of the chip's functions. The driving electrodes 2 are arranged on the PCB substrate 1 and are used for the loading, transportation, mixing, and liquid separation operations of the PCR reaction liquid (template + PCR mix) to be manipulated on the PCB substrate. The dielectric water - transporting layer includes a top layer 4 and a bottom layer 3. The top layer 4 and the bottom layer 3 are fitted and fixed to encapsulate and seal the sealing oil 6. The sealing oil 6 is a highly stable hydrophobic insulating oil, and both the sealing oil 6 and the droplets 7 are injected or extracted through the sampling and injection holes 9 opened on the upper cover plate 5 and the top layer 4.

[0040] Among them, the driving electrode 2 has a liquid - carrying volume of a single electrode of 3 μL, and the combined control of the 6 - μL droplet 7 volume can be achieved through matrix splicing.

[0041] It should be noted that in addition to the PCB material, the PCB substrate 1 can select other materials with low cost, easy processing, and having chemical stability and mechanical strength, such as polymer materials (such as polydimethylsiloxane PDMS) or composite materials. These materials can also reduce costs and are suitable for large - scale production, and at the same time have a certain degree of flexibility in processing and function realization; the sealing effect of silicone oil can be replaced by other low - volatility and chemically stable liquids, such as fluorinated oil or other synthetic oils. These media can also provide excellent sealing effects and reduce the pollution risk during chip operation.

[0042] Embodiment 2

[0043] As Figure 3-4 shown, the partition of the multi - sample parallel two - step PCR digital microfluidic chip includes:

[0044] A sample loading area 11 for initial sample introduction, a mixing and liquid separation area 22 for precise liquid separation and mixing of liquids, a reaction area 33 provided with an independent temperature control unit, a liquid injection and collection area 44, an oil - seal closed area 55, and a conductive area 66 for realizing the conduction of the driving electrode 2 to achieve the electro - wetting effect.

[0045] The sample loading area 11 adopts a matrix electrode arrangement mode, which can achieve efficient mixing of multi-reagent microdroplets. Specifically, the sample loading area 11 is divided into a cold loading area 111 and a hot loading area 112. The liquid injection and collection area 44 includes a cold loading hole 441 and a hot loading hole 442 corresponding to the cold loading area 111 and the hot loading area 112, and also includes a sample hole 443 for final inspection liquid collection and processing;

[0046] Specifically, the triangular electrode structure in the mixing and dispensing area 22 is controlled by logic electrodes to achieve right-angle dispensing to ensure the accuracy and uniformity of the dispensing process;

[0047] The reaction area 33 provides accurate temperature control through an independent temperature control unit to meet the reaction requirements of two-step PCR. Specifically, the reaction area 33 includes a high-temperature area 331 and a low-temperature area 332 that meet the reaction requirements of two-step PCR, and controls the switching of the droplet 7 between the high-temperature area 331 and the low-temperature area 332 through electrowetting path planning;

[0048] The liquid injection and collection area 44 is used to inject and store the reaction solution, and can also collect the final inspection liquid for convenient subsequent processing or verification;

[0049] The oil seal and sealing area 55 connects the upper cover plate 5 and the PCB substrate 1, is used to encapsulate and seal the oil 6, and prevents the liquid from contacting the outside world.

[0050] Before conducting the experiment, the sealing oil 6 can be injected through the cold loading hole 441 or the hot loading hole 442, and the liquid is added to the sample loading area 11 to meet the different temperature loading requirements of different experiments. Through matrix splicing, various complex dispensing and mixing operations can be customized. Subsequently, according to the designed volume of 3 ul per single electrode, the required liquid ratio and volume can be reasonably divided by relying on matrix observation without using a measuring tool. Usually, to avoid false positives in the experiment, the PCR reaction liquid is required to be greater than 5 ul. Therefore, it is ensured that the volume of the droplet 7 is 2 electrodes, that is, 6 ul. The sample loading area 11 provides a large enough bearing space, and precise sample loading is not required, only the target reaction volume and sample ratio need to be ensured;

[0051] After the sample pretreatment is completed, a liquid dispensing electrode (i.e., after completing the above procedures) is required. Right-angle dispensing is achieved by relying on the triangular electrode structure in the mixing and dispensing area 22 to achieve multi-target precise dispensing. The remaining control samples that do not need to be processed can be directly injected through the sample hole 443 and driven to the target position. The PCR amplification of the liquid is carried out in the reaction area 33. In the two-step PCR experiment content, through the set high-temperature area 331 and low-temperature area 332, the target liquid can achieve temperature zone switching in the two areas through liquid drive path planning;

[0052] After the reaction is completed, the final inspection solution can be collected through the sample hole 443 according to the procedure (it can be replaced by other driving methods, such as thermocapillary driving, optical driving or acoustic driving. These methods can also achieve precise manipulation of the droplet 7 and may have higher efficiency or stability in certain specific scenarios).

[0053] This experimental operation can be carried out manually at the GUI (user interface), or through an automated control system (that is, the above steps are completed automatically), and by adjusting the temperature control and fluid distribution in the chip in real time, support for more complex biological reactions can be achieved.

[0054] Figure 1 The electrode is in a non-conductive state, while Figure 2 The electrode is in a conductive state. Its electrode array is reasonably arranged according to the sample embodiment, and the loading, mixing, transmission and reaction operations of the microdroplets are realized through precise voltage and ingenious logic control.

[0055] It should be noted that the structure described in the present invention can be implemented in many different forms and is not limited to the embodiments. Any equivalent transformation made by those of ordinary skill in the art using the specification and drawings of the present invention, or directly or indirectly applied in other related technical fields, such as the loading and unloading of other items, is included in the protection scope of the present invention.

Claims

1. A multi-sample parallel two-step PCR digital microfluidic chip, characterized by: It includes a PCB substrate, a driving electrode, a dielectric water transfer layer and an upper cover plate which are sequentially distributed from bottom to top, wherein the PCB substrate and the upper cover plate are sealed by glue, and the dielectric water transfer layer is filled with sealing oil, and the sealing oil contains immiscible droplets; The PCB substrate is provided with an electrode circuit for realizing the electrowetting effect; The driving electrodes are arranged on the PCB substrate and are used to control the loading, transmission, mixing and liquid separation operations of the sample liquid; The dielectric water transfer layer comprises a top layer and a bottom layer, the top layer and the bottom layer are attached and fixed to each other and wrap and seal the sealing oil; The sealing oil and liquid droplets are injected or extracted through the sampling holes opened on the upper cover plate and the top layer.

2. The multi-sample parallel two-step PCR digital microfluidic chip according to claim 1, characterized in that: The driving electrode is a single electrode with a liquid carrying volume of 3 μL.

3. The multi-sample parallel two-step PCR digital microfluidic chip according to claim 1, characterized in that: The sealing oil is a highly stable hydrophobic insulating oil.

4. An electrode layout of a multi-sample parallel two-step PCR digital microfluidic chip, characterized in that: The multi-sample parallel two-step PCR digital microfluidic chip according to any one of claim 13 further comprises the following partitions: a sample loading area for initial sample introduction; A mixing and dispensing area for precise dispensing and mixing of liquids; A reaction zone is provided with an independent temperature control unit for adjusting the temperature; A liquid injection collection area for storing liquid after injection and collecting and processing final test liquid; Oil seal enclosed area used to encapsulate sealing oil and prevent liquid from contacting the outside world; Conductive area used to achieve electrode conduction and electrowetting effect.

5. The electrode layout of a multi-sample parallel two-step PCR digital microfluidic chip as claimed in claim 4, characterized in that: The sample loading area is divided into a cold loading area and a hot loading area. The liquid injection collection area includes cold loading holes and hot loading holes corresponding to the cold loading area and the hot loading area, and also includes a sample hole for collecting and processing the final test liquid.

6. The electrode layout of a multi-sample parallel two-step PCR digital microfluidic chip as claimed in claim 4, characterized in that: The triangular electrode structure of the mixing and liquid separation area is controlled by a logic electrode.

7. The electrode layout of a multi-sample parallel two-step PCR digital microfluidic chip as claimed in claim 4, characterized in that: The reaction zone includes a high temperature zone and a low temperature zone that meet the requirements of a two-step PCR reaction, and the droplets are controlled to switch between the high temperature zone and the low temperature zone through electrowetting path planning.

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