A multi-sample parallel two-step method PCR digital microfluidic chip and electrode layout

Abstract

The application discloses a kind of multi-sample parallel two-step method PCR digital microfluidic chip and electrode layout, chip includes by sequentially distributed from bottom to top PCB substrate, driving electrode, dielectric water transport layer and upper cover, the PCB substrate is sealed with upper cover by glue, and dielectric water transport layer is filled with sealing oil, the sealing oil is wrapped with immiscible droplet, electrode layout includes loading area, mixed liquid separation area, reaction area, liquid injection collection area, oil seal closed area and conductive area;Chip uses PCB material as the basis of microfluidic chip, not only greatly reduces production cost, and makes chip more easily realize mass production and application, electrode layout is through optimizing electrode arrangement, ensure that microdroplet can be accurately, efficiently moved and operated in chip, significantly reduce the complexity of design and control, for the requirement of two-step method PCR design, not only can realize the efficient transmission, mixing and separation of sample, also can ensure the precision and efficiency of reaction.

Description

TECHNICAL FIELD

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

[0002] Digital microfluidics (DMF) is a microfluidic technology that precisely manipulates small droplets through electrowetting on dielectric (EWOD) effect. Unlike traditional continuous flow microfluidics, digital microfluidics can control the generation, splitting, merging, and transport of droplets on an open two-dimensional chip surface by applying electric fields. Due to its high flexibility and precise droplet control capability, 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 diagnostics. Traditional PCR includes three main steps: denaturation, annealing, and extension. Two-step PCR can be used when rapid amplification or simplified operation process is required. 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 transitions, 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 leverage the advantages of both, achieving efficient nucleic acid amplification. First, through digital microfluidic technology, samples and PCR reaction mixtures are precisely dispensed into multiple independent droplets on the chip, each droplet serving as an independent reaction unit for processing. In the first stage, the DNA sample in the droplet will be denatured at a high temperature zone, causing double-stranded DNA to uncoil into single strands, preparing for amplification. Subsequently, in the second stage, the droplet moves to a low temperature zone, both annealing (primer binding to target DNA) and extension (DNA polymerase synthesizing new DNA strands) are performed. The advantage of digital microfluidic systems is that multiple droplets can be flexibly controlled to react simultaneously through an electrode array, achieving multi-sample parallel processing, and each droplet is independent of each other, greatly reducing the risk of cross-contamination. The entire process can be fully automated, greatly improving the efficiency and simplicity of the experiment.

[0005] The conventional digital microfluidic chip mainly uses several materials such as silicon and glass in material selection. The silicon material is often used in applications requiring high-precision microfluidic structure and temperature control system due to its excellent thermal conductivity, chemical stability and mechanical strength. However, the silicon material has high cost and complex manufacturing process, and is not suitable for low-cost mass production. The glass has excellent optical transparency and chemical stability, but its processing difficulty and cost are also high. The application provides a novel multi-sample parallel two-step PCR digital microfluidic chip.

[0006] The electrode layout in the prior art has many deficiencies in design. First, the electrode distribution is usually not uniform enough to cover the entire microdroplet movement area, which limits the movement of the microdroplets and makes it difficult to achieve efficient sample transmission and mixing. In addition, the electrode driving circuit of the existing layout often needs complex control logic, which is difficult to operate and is prone to misoperation, affecting the stability of the experimental results. More importantly, the existing layout is designed for a single process, lacks adaptability, and is difficult to meet the complex requirements of multi-stage temperature control and reagent distribution in two-step PCR. SUMMARY

[0007] In view of the above defects, the application provides a multi-sample parallel two-step PCR digital microfluidic chip, which comprises a PCB substrate, a driving electrode, a dielectric water transport layer and an upper cover plate distributed in turn from bottom to top, the PCB substrate and the upper cover plate are sealed by glue, and the dielectric water transport layer is filled with sealing oil, and the sealing oil is wrapped with immiscible droplets;

[0008] The PCB substrate is provided with an electrode circuit for realizing the electrowetting effect;

[0009] The driving electrode is arranged on the PCB substrate and is used for controlling the loading, transmission, mixing and dispensing operation of the sample liquid;

[0010] The dielectric water transport layer comprises a top layer and a bottom layer, the top layer and the bottom layer are fixedly attached and seal the sealing oil;

[0011] The sealing oil and the droplets are injected or extracted through the sampling holes opened in the upper cover plate and the top layer.

[0012] Further, the driving electrode is a single electrode with a liquid carrying volume of 3 mu L.

[0013] Further, the sealing oil is a high-stability hydrophobic insulating oil.

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

[0015] A sample loading area for initial sample introduction;

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

[0017] A reaction area provided with an independent temperature control unit for temperature adjustment;

[0018] A liquid injection and collection area for post-injection storage and final inspection liquid collection and processing;

[0019] An oil seal area for sealing and avoiding contact of liquids with the outside world;

[0020] An electrically conductive area for realizing electrode conduction and achieving electrowetting 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 final inspection liquid collection and processing.

[0022] Further, the triangular electrode structure of the mixing and dispensing area is controlled by a logic electrode to realize 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 to meet the requirements of two-step PCR reaction, and the switching of droplets between the high-temperature area and the low-temperature area is controlled through an electrowetting path planning.

[0024] Compared with the prior art, the present application has the following beneficial effects:

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

[0026] 2. By optimizing the electrode arrangement layout, the microdroplets can be accurately and efficiently moved and operated in the chip, significantly reducing the design and control complexity, and the design is specially designed for two-step PCR requirements, which can not only realize 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 application particularly supports parallel processing of multiple samples, which can simultaneously carry out multiple independent PCR experiments on a single chip, thereby significantly improving the experimental throughput and work efficiency, at the same time, reasonable division of electrode area and optimized driving algorithm effectively reduce the energy consumption of the chip, and improve the running stability and applicability of the system;

[0028] 4、Through optimizing the chip structure and electrode layout design, combining with the electrowetting effect and path planning, the multi-sample parallel two-step PCR reaction operation is successfully realized, which has the characteristics of high efficiency, precision and modularization, and has wide application potential in medical detection, scientific research experiment and portable molecular diagnosis equipment. BRIEF DESCRIPTION OF DRAWINGS

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

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

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

[0032] Figure 4 It is a detailed schematic diagram of Figure 3 .

[0033] Figure 5 It is a PCB design diagram of the 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 hole; 11, sample loading area; 111, cold sample loading area; 112, hot sample loading area; 22, mixing and separation area; 33, reaction area; 331, high temperature area; 332, low temperature area; 44, liquid injection and collection area; 441, cold sample loading hole; 442, hot sample loading hole; 443, sample hole; 55, oil seal closed area; 66, conductive area. DETAILED DESCRIPTION

[0035] In order to facilitate the understanding of the present application, the device of the present application will be described more fully below with reference to the related drawings. Embodiments of the device are shown in the drawings. However, the device can be realized in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present application more thorough and comprehensive.

[0036] In the description of the present application, it should be noted that unless otherwise explicitly specified and limited, the terms "mounting", "connection", "setting" should be understood broadly, for example, it can be fixedly connected, set, or detachably connected, set, or integrally connected, set. For those skilled in the art, the specific meaning of the above terms in the present application can be understood according to the specific circumstances.

[0037] Example 1

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

[0039] In detail, the PCB substrate 1 is configured with an electrode circuit for realizing the electrowetting effect, ensuring efficient operation of the chip function, the driving electrode 2 is arranged on the PCB substrate 1, used for loading, transporting, mixing and separating the manipulated PCR reaction liquid (template + PCR mix) on the PCB substrate, the dielectric water transport layer includes a top layer 4 and a bottom layer 3, the top layer 4 and the bottom layer 3 are fixedly attached and wrapped with the sealing oil 6, the sealing oil 6 is a high-stability hydrophobic insulating oil, and the sealing oil 6 and the droplets 7 are injected or extracted through the sampling hole 9 opened in the upper cover plate 5 and the top layer 4.

[0040] Among them, the driving electrode 2 is a single electrode with a liquid carrying volume of 3μL, and the combination control of 6μL droplets 7 volume can be realized by matrix splicing.

[0041] It should be noted that in addition to the PCB material, the PCB substrate 1 can also select other low-cost, easy-to-process and chemically stable and mechanically strong materials, such as polymer materials (such as polydimethylsiloxane PDMS) or composite materials. These materials can also reduce costs and be suitable for mass production, and have certain flexibility in processing and function realization; the sealing effect of silicone oil can be replaced by other low-volatility, chemically stable liquids, such as fluorine oil or other synthetic oils. These media can also provide excellent sealing effect and reduce the risk of pollution during chip operation.

[0042] Embodiment 2

[0043] As Figures 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 separating area 22 for precise liquid separation and mixing, a reaction area 33 provided with an independent temperature control unit, a liquid injection and collection area 44, an oil seal area 55, and a conductive area 66 for realizing the conduction of the driving electrode 2 to realize the electrowetting effect.

[0045] The sample loading area 11 adopts a matrix electrode arrangement mode, and can realize efficient mixing of multiple reagent droplets. In detail, 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 further includes a sample hole 443 for collecting and processing the final detection liquid;

[0046] In detail, the triangular electrode structure of the mixing and separation area 22 is controlled by a logic electrode to realize right-angle separation to ensure the accuracy and uniformity of the separation 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. In detail, the reaction area 33 includes a high-temperature area 331 and a low-temperature area 332 to meet the reaction requirements of two-step PCR, and the droplet 7 is switched between the high-temperature area 331 or the low-temperature area 332 through the liquid driving path planning control.

[0048] The liquid injection and collection area 44 is used for injecting and storing reaction liquid, and can also collect the final detection liquid for subsequent processing or verification.

[0049] The oil seal area 55 is connected to the upper cover plate 5 and the PCB substrate 1, and is used for packaging and sealing the oil 6, and avoiding contact between the liquid and the outside.

[0050] Before the experiment, the oil 6 can be injected through the cold loading hole 441 or the hot loading hole 442, and the liquid can be added in the sample loading area 11 to meet the different temperature loading requirements of different experiments. Through matrix splicing, various complex separation and mixing work can be realized. According to the design volume of the single electrode 3ul, the required liquid proportion and volume can be reasonably divided in the form of matrix observation, which usually avoids false positive in experiments and requires PCR reaction liquid to be greater than 5ul. Therefore, the volume of the droplet 7 is 6ul, which is 2 electrodes, the sample loading area 11 provides a large enough bearing space, and does not need to be accurately loaded, but only needs to ensure the target volume and sample proportion.

[0051] After the sample pretreatment is completed, the liquid separation electrode (i.e. the above-mentioned process is completed), the triangular electrode structure at the mixing and separation area 22 realizes right-angle separation to realize precise separation of multiple targets, and the remaining samples without treatment can be directly injected and driven to the target position through the sample hole 443. The PCR amplification of the liquid is carried out in the reaction area 33. In the two-step PCR experiment, through the high-temperature area 331 and the low-temperature area 332, the target liquid can be switched between the two areas through the liquid driving path planning.

[0052] After the reaction is complete, the final test liquid can be collected through the sample well 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 control of droplets 7, and may have higher efficiency or stability in certain specific scenarios).

[0053] This experiment can be performed manually via the GUI (user interface) or through an automated control system (i.e., automating the above steps). By adjusting the temperature control and fluid distribution within the chip in real time, it can support more complex biological reactions.

[0054] Figure 1 When the electrode is in a non-conductive state, and Figure 2 With the electrodes in the conducting state, the electrode array is rationally arranged according to the sample embodiment, and the loading, mixing, transport and reaction operations of microdroplets are realized through precise voltage and ingenious logic control.

[0055] It should be noted that the structure described in this invention can be implemented in many different forms and is not limited to the embodiments described. Any equivalent transformations made by those skilled in the art based on the description and drawings of this invention, or direct or indirect applications in other related technical fields, such as the loading and unloading of other items, are included within the protection scope of this invention.

Claims

1. A digital microfluidic chip for multi-sample parallel two-step PCR, characterized in that: The device includes a PCB substrate, a driving electrode, a dielectric water transport layer, and a top cover plate arranged from bottom to top. The PCB substrate and the top cover plate are sealed with adhesive, and the dielectric water transport layer is filled with sealing oil, which contains immiscible droplets. The PCB substrate is equipped with electrode lines for realizing the electrowetting effect; The driving electrode is arranged on the PCB substrate. The driving electrode has a single electrode volume of 3μL and is used to control the loading, transfer, mixing and separation of sample liquid. The dielectric water-conducting layer includes a top layer and a bottom layer, which are bonded and fixed together and sealed with sealing oil. Both the sealing oil and the droplets are injected or extracted through sampling holes provided in the upper cover plate and the top layer. It also includes a mixing and dispensing zone for precise liquid separation and mixing. The triangular electrode structure of the mixing and dispensing zone is controlled by logic electrodes to achieve right-angle dispensing to ensure the accuracy and uniformity of the dispensing process.

2. The digital microfluidic chip for multi-sample parallel two-step PCR as described in claim 1, characterized in that: The sealing oil is a highly stable hydrophobic insulating oil.

3. An electrode layout for a multi-sample parallel two-step PCR digital microfluidic chip, characterized in that, The digital microfluidic chip for multi-sample parallel two-step PCR as described in any one of claims 1-2 further includes the following partitions: Sample loading area used for initial sample introduction; A mixing and separating zone for precise separation and mixing of liquids; The reaction zone is equipped with an independent temperature control unit for temperature adjustment; Liquid injection collection area for post-injection storage and final test liquid collection and treatment; An oil seal is a sealed area used to encapsulate sealing oil and prevent the liquid from contacting the outside environment; The conductive region used to achieve electrowetting effect by enabling electrode conduction.

4. The electrode layout of the digital microfluidic chip for multi-sample parallel two-step PCR as described in claim 3, characterized in that: 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 final test liquid collection and processing.

5. The electrode layout of the digital microfluidic chip for multi-sample parallel two-step PCR as described in claim 3, characterized in that: The reaction zone includes a high-temperature zone and a low-temperature zone to meet the requirements of the two-step PCR reaction, and the droplets are switched between the high-temperature zone and the low-temperature zone through electrowetting path planning.

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