Digital PCR microfluidic chip

Abstract

The utility model discloses a kind of digital PCR microfluidic chip, including sequentially bonded from top to bottom micropore layer, channel layer and sealing layer, sample inlet rubber plug and sample outlet rubber plug;Micropore layer top surface one end is fixed with sample inlet cup, and the other end is fixed with sample outlet cup;The cup mouth of sample inlet cup and sample outlet cup is respectively plugged by sample inlet rubber plug and sample outlet rubber plug;The bottom surface of micropore layer is equipped with micropore array area and its both ends respectively communicate the cup mouth of sample inlet cup and sample outlet cup;Reagent flow channel corresponding micropore array area is opened on channel layer;Sealing layer is equipped with waste liquid tank on the top surface corresponding sample outlet cup.The utility model adds sample cup to reagent flow channel in succession by adding PCR amplification reagent and sealing oil, presses sample inlet rubber plug, and pushes reagent and sealing oil to advance in reagent flow channel, completes the drop of reagent and the drop separation of sealing oil, and finally presses sample outlet rubber plug to realize the sealing of entire chip;The chip is simple to operate, does not need additional driving, pressurizing equipment, and can realize digitization and amplification.

Description

Technical Field

[0001] This utility model relates to the field of nucleic acid detection technology, and more specifically to a digital PCR microfluidic chip. Background Technology

[0002] Digital PCR (dPCR) is a novel method for absolute quantitative nucleic acid detection. It is highly adaptable to complex samples, exhibits high detection sensitivity, and has broad application prospects in various fields such as medicine and biology. Compared to traditional PCR, digital PCR adds a crucial step—reagent system distribution. This involves dispersing tens of microliters of nucleic acid into thousands to millions of independent microreaction units, ensuring that each unit contains either zero or one DNA template molecule. These thousands or tens of thousands of independent reaction units are simultaneously subjected to PCR amplification. After amplification, the fluorescence signals of each reaction unit are statistically analyzed, thereby achieving absolute quantification of the target molecule.

[0003] Depending on the distribution method, dPCR is mainly divided into two categories: droplet-based dPCR and chip-based dPCR. Droplet-based dPCR uses microfluidic technology to distribute the sample into tens of thousands of water-in-oil droplets, each droplet acting as an independent PCR reaction unit. It offers high flexibility, allowing for adjustments to droplet size or number to meet different throughput requirements. However, it also places high demands on the stability and uniformity of the generated droplets. Chip-based dPCR, on the other hand, uses a microarray chip to disperse the sample into tens of thousands of wells, each well acting as an independent PCR reaction unit. This distribution method offers superior microsystem size, stability, and uniformity of arrangement compared to droplet-based dPCR, and is therefore widely used in research and clinical applications.

[0004] Current chip-based digital PCR still has the following problems:

[0005] 1. Existing methods all involve two steps of adding liquid and oil to distribute the system, which requires repeated plugging and unplugging for transfer or manipulation using specialized equipment. Therefore, from chip fabrication to amplification and detection, multiple devices are usually required, making the operation cumbersome, inefficient, and prone to cross-contamination.

[0006] 2. Common materials for digital PCR chips include PDMS and silicon. PDMS is prone to sample loss and aerosol contamination due to insufficient chip sealing because of its permeability. Silicon-based chips are complex to manufacture and expensive, making them unsuitable for mass production.

[0007] 3. To achieve the coverage of the microwells in the chip, the volume of the system added to the chip is usually greater than the total volume of the microwells. After the distribution is completed, the remaining dPCR samples must be completely isolated from the valid sample wells or manually excluded. Otherwise, the amplification system is easily contaminated during the amplification process, which is cumbersome and difficult to automate.

[0008] 4. To avoid the impact of evaporation of the microsystems generated within such microfluidic chips on detection during amplification, patents CN202010232789.9 and CN201910764194.5 employ methods such as providing additional pressurization devices to the amplification chamber to prevent evaporation. However, these methods result in complex instrument configurations and significant development challenges.

[0009] Digital PCR technology is widely believed to make up for the shortcomings of conventional PCR in absolute quantification, bringing new breakthroughs to clinical testing and treatment, and having great significance for life and health. However, the above-mentioned aspects have always hindered the application of digital PCR.

[0010] Therefore, how to provide a device-driver-free, fully sealed digital PCR microfluidic chip is a problem that urgently needs to be solved by those skilled in the art. Utility Model Content

[0011] In view of this, the present invention provides a digital PCR microfluidic chip, in which PCR amplification reagent and sealing oil are added sequentially at the sample dispensing port, and a disposable rubber stopper is pressed to push the reagent and sealing oil forward in the chip channel, completing the separation of reagent droplets and sealing oil droplets, and finally the rubber stopper seals the entire chip; its operation is simple and does not require additional driving or pressurizing equipment to achieve digitization and amplification.

[0012] To achieve the above objectives, the present invention adopts the following technical solution:

[0013] A digital PCR microfluidic chip includes, from top to bottom, a microporous layer, a channel layer, a sealing layer, an injection plug, and an outlet plug bonded together.

[0014] A sample inlet cup is fixed at one end of the top surface of the microporous layer, and a sample outlet cup is fixed at the other end; the openings of the sample inlet cup and the sample outlet cup are respectively sealed by the sample inlet stopper and the sample outlet stopper; the bottom surface of the microporous layer is provided with a microporous array area and its two ends are respectively connected to the openings of the sample inlet cup and the sample outlet cup;

[0015] The channel layer has reagent channels corresponding to the micropore array area; the top surface of the sealing layer has a waste liquid tank corresponding to the sample cup.

[0016] The beneficial effects of this utility model are that PCR amplification reagent and sealing oil are added to the reagent channel in sequence by adding the sample cup, pressing the sample plug, pushing the reagent and sealing oil forward in the reagent channel, completing the separation of the reagent droplet and the sealing oil droplet, and finally pressing the sample outlet plug to seal the entire chip; the chip is simple to operate and can realize digitization and amplification without the need for additional driving or pressurizing equipment.

[0017] Preferably, the sample inlet is provided on the side wall of the sample inlet cup, and the sample inlet stopper can slide along the inner cavity of the sample inlet cup to close the sample inlet. The sample inlet stopper is positioned above the sample inlet, and reagent is injected through the sample inlet. Pressing the sample inlet stopper squeezes the reagent, causing it to flow along the direction of the sample outlet cup.

[0018] Preferably, the sample dispensing cup has vent holes on its side wall, and the sample dispensing stopper can slide along the inner cavity of the sample dispensing cup to seal the vent holes. Before use, the sample dispensing stopper is positioned above the vent holes. After the reagent fills the microarray area, the sample dispensing stopper is pressed to seal the vent holes. At this time, the chip is completely sealed, with no excess gas causing disturbance and no aerosol contamination. The chip is then placed on a PCR amplification platform, and the amplification program is started to achieve digital PCR amplification.

[0019] Preferably, the microwell array region consists of multiple equally sized hexagonal, circular, or quadrilateral microwell arrays. This ensures the accuracy and validity of the digital PCR amplification results.

[0020] Preferably, both the inner and outer surfaces of the micropores in the micropore array are coated with a hydrophilic coating or a hydrophilic reagent. Utilizing the hydrophilicity of the micropore array, the reagent can self-propelledly fill the micropores sequentially.

[0021] Preferably, the hydrophilic coating is one or more of the following: Plasma, polymer hydrophilic coating, nano-hydrophilic coating, and hydrophilic grafting.

[0022] Preferably, the hydrophilic reagent is bonded to the inner or outer surface of the micropores by vapor deposition, vapor grafting, spraying, or impregnation.

[0023] Preferably, the channel layer is composed of one or more of PC, COC, COP, PET, and double-sided adhesive.

[0024] Preferably, the microporous layer and the sealing layer are composed of one or more of PC, COC, COP, and PET.

[0025] Preferably, the microporous layer, channel layer, and sealing layer are bonded together by adhesive bonding, thermo-press bonding, or solvent bonding.

[0026] As can be seen from the above technical solution, compared with the prior art, the present invention discloses a digital PCR microfluidic chip, which has the following beneficial effects:

[0027] 1. No equipment is required to drive the reagents and oil phase into the chip, and there is no need to repeatedly plug and unplug reagent tubes and oil tubes. The reagent distribution and oil phase isolation and sealing are completed manually by adding sample and oil at one time, along with the sample inlet rubber stopper and sample outlet rubber stopper.

[0028] 2. The chip is designed with a waste liquid tank structure. The remaining reagents fall into the waste liquid tank at the outlet and are completely isolated from the effective sample well by an oil seal to avoid sample contamination caused by disturbances during the amplification process.

[0029] 3. No additional pressure device is required at the instrument end. The chip is fully sealed by the cooperation of the sample inlet and outlet rubber plugs at both ends, avoiding reagent evaporation during amplification and sample contamination. Attached Figure Description

[0030] To more clearly illustrate the technical solutions in the embodiments of this utility model 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 embodiments of this utility model. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0031] Figure 1 This is a schematic diagram of the chip's exploded structure provided by this utility model;

[0032] Figure 2 A schematic diagram of the back structure of the microporous layer provided by this utility model;

[0033] Figure 3 A schematic diagram of the chip operation process provided by this utility model;

[0034] Figure 4 This is a schematic diagram of the operation process of Embodiment 3 provided by this utility model;

[0035] Figure 5 This is a schematic diagram of the operation process of Embodiment 4 provided by this utility model.

[0036] in,

[0037] 1-Microwell layer; 11-Microwell array area; 12-Microwell array; 2-Channel layer; 21-Reagent flow channel; 3-Sealing layer; 31-Waste liquid tank; 4-Sample cup; 41-Sample inlet; 5-Sample outlet cup; 51-Vent; 6-Sample stopper; 7-Sample outlet stopper; 8-Injection piston; 9-Amplification chamber; 10-Spike array structure. Detailed Implementation

[0038] The technical solutions of the present utility model will be clearly and completely described below with reference to the accompanying drawings of the embodiments. Obviously, the described embodiments are only some embodiments of the present utility model, and not all embodiments. Based on the embodiments of the present utility model, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the protection scope of the present utility model.

[0039] Example 1:

[0040] See appendix Figures 1-2 This utility model discloses a digital PCR microfluidic chip, which includes a microporous layer 1, a channel layer 2, a sealing layer 3, an injection plug 6, and an outlet plug 7 bonded from top to bottom.

[0041] A sample inlet cup 4 is fixed at one end of the top surface of the microporous layer 1, and a sample outlet cup 5 is fixed at the other end. The openings of the sample inlet cup 4 and the sample outlet cup 5 are sealed by the sample inlet stopper 6 and the sample outlet stopper 7, respectively. A microporous array region 11 is provided on the bottom surface of the microporous layer 1, and its two ends are connected to the openings of the sample inlet cup 4 and the sample outlet cup 5, respectively.

[0042] The channel layer 2 has a reagent flow channel 21 corresponding to the micropore array area 11; the top surface of the sealing layer 3 has a waste liquid tank 31 corresponding to the sample cup 5.

[0043] In this embodiment, the microporous layer 1, the channel layer 2, and the sealing layer 3 are bonded together by adhesive bonding, thermoforming bonding, or solvent bonding. Regardless of the bonding method used, it is necessary to ensure the sealing performance between the layers.

[0044] To further optimize the above technical solution, the microporous layer 1 and the sealing layer 3 are composed of one or more of PC, COC, COP, and PET. The channel layer 2 is composed of one or more of PC, COC, COP, PET, and double-sided adhesive.

[0045] To further optimize the above technical solution and prevent cross-contamination during the testing process, both the injection stopper and the outlet stopper are disposable.

[0046] In some other specific embodiments, to ensure the accuracy of the test structure, the micropore array region 11 includes, but is not limited to, a micropore array 12 consisting of multiple equally sized hexagonal, circular, or quadrilateral structures.

[0047] To further optimize the above technical solution and achieve self-driven movement of the reagent, the inner and outer surfaces of the micropores on the micropore array 12 are coated with a hydrophilic coating or a hydrophilic reagent for hydrophilic treatment.

[0048] When a hydrophilic coating is used, the hydrophilic coating can be one or a combination of plasma, polymer hydrophilic coating, nano-hydrophilic coating, or hydrophilic grafting. When a hydrophilic reagent is used, the hydrophilic reagent is bonded to the inner or outer surface of the micropores through vapor deposition, vaporization grafting, spraying, or impregnation.

[0049] In this embodiment, the side wall of the injection cup 4 is provided with an injection port 41, and the injection stopper 6 can slide along the inner cavity of the injection cup 4 to close the injection port 41. The side wall of the discharge cup 5 is provided with an air hole 51, and the discharge stopper 7 can slide along the inner cavity of the discharge cup 5 to close the air hole 51.

[0050] In this embodiment, there are four reagent channels, which do not interfere with each other and correspond to four injection cups and four output cups respectively.

[0051] like Figure 3 As shown, when performing digital PCR amplification using a digital PCR microfluidic chip, the advance and exit stoppers are pre-assembled on the microfluidic chip. The specific steps include:

[0052] S1. Add digital PCR reagent and sealing oil sequentially to the chip inlet. The digital PCR reagent is driven forward and sequentially fills the microwells through the capillary action of the reagent channels on the channel layer and the hydrophilic action of the microwell array area.

[0053] S2. Manually press the injection stopper. The air between the sealing oil and the injection stopper is expelled from the chip through the injection port until the injection stopper seals the injection port. Continue pressing the injection stopper to push the sealing oil in the injection cup into the channel. The reagent in the channel falls further forward into the hole. At the same time, the sealing oil seals the reagent that has been effectively dropped into the hole at the front end in the micropore to form an independent small droplet.

[0054] S3. After the reagent is effectively dropped into the well, the excess reagent is pushed into the waste liquid tank and sealed with sealing oil, completely isolating it from the front sample well to prevent the front sample from being contaminated by disturbance during the amplification process.

[0055] S4. After all reagents have been dispensed, press the injection stopper further to allow the sealing oil to enter the sample cup and expel the air from the sample cup through the vent.

[0056] S5. Press the sample dispensing stopper. The air between the sample dispensing stopper and the sealing oil will be discharged through the air hole until the sample dispensing stopper seals the air hole.

[0057] After step S5 is completed, the chip is completely sealed inside, with no excess gas causing disturbance and no aerosol contamination. Place the chip on the PCR amplification platform, start the amplification program, and digital PCR amplification can be achieved.

[0058] Example 2:

[0059] According to the appendix Figure 4 This utility model discloses an alternative solution for digital PCR nucleic acid detection. The injection piston 8, filled with sample reagents and oil phase, corresponds to the sample inlet cup 4 of the microfluidic chip. The injection piston 8 is manually pressed down to inject reagents into the sample inlet cup to realize the addition of sample and oil phase and the distribution of sample, thus completing the preparation of digital PCR chip.

[0060] After removing the injection piston, place the digital PCR chip inside the amplification instrument. The two ends of the chip are exposed in the amplification chamber 9 inside the amplification instrument. During amplification, an external pressure device applies air pressure to the amplification chamber 13 to prevent the evaporation of reagents in the wells inside the chip during the amplification process.

[0061] Example 3:

[0062] According to the appendix Figure 5 The digital PCR microfluidic chip of this utility model solves the problem of air bubbles appearing during the amplification process.

[0063] During amplification, the microfluidic chip is fixed on the tilted amplification platform inside the amplification instrument. The bottom of the chip remains parallel and tightly attached to the amplification platform. Due to the low density of bubbles, the bubbles that appear during amplification move to the higher side of the tilted chip, pass through the oil phase, and reach the sample cup 4. The spike array structure 10 at the bottom of the sample cup 4 punctures the bubbles, promptly expelling and breaking them, thus avoiding interference with the amplification and detection process.

[0064] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on its differences from other embodiments. Similar or identical parts between embodiments can be referred to interchangeably. For the apparatus disclosed in the embodiments, since they correspond to the methods disclosed in the embodiments, the description is relatively simple; relevant parts can be referred to the method section.

[0065] The above description of the disclosed embodiments enables those skilled in the art to make 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 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. A digital PCR microfluidic chip, characterized in that, It includes a microporous layer (1), a channel layer (2), a sealing layer (3), an injection plug (6), and an outlet plug (7) bonded sequentially from top to bottom; A sample inlet cup (4) is fixed at one end of the top surface of the microporous layer (1), and a sample outlet cup (5) is fixed at the other end; the openings of the sample inlet cup (4) and the sample outlet cup (5) are sealed by the sample inlet stopper (6) and the sample outlet stopper (7) respectively; the bottom surface of the microporous layer (1) is provided with a microporous array area (11) and its two ends are respectively connected to the openings of the sample inlet cup (4) and the sample outlet cup (5); The channel layer (2) is provided with a reagent flow channel (21) corresponding to the micropore array region (11); the top surface of the sealing layer (3) is provided with a waste liquid tank (31) corresponding to the sample cup (5).

2. The digital PCR microfluidic chip according to claim 1, characterized in that, The side wall of the injection cup (4) is provided with an injection port (41), and the injection rubber stopper (6) can slide along the inner cavity of the injection cup (4) to close the injection port (41).

3. The digital PCR microfluidic chip according to claim 1, characterized in that, The side wall of the sample cup (5) is provided with an air hole (51), and the sample plug (7) can slide along the inner cavity of the sample cup (5) to seal the air hole (51).

4. The digital PCR microfluidic chip according to claim 1, characterized in that, The micropore array region (11) is composed of multiple equally sized hexagonal, circular or quadrilateral micropore arrays (12).

5. A digital PCR microfluidic chip according to claim 4, characterized in that, The inner and outer surfaces of the micropores in the micropore array (12) are coated with a hydrophilic coating or a hydrophilic reagent.

6. A digital PCR microfluidic chip according to claim 5, characterized in that, The hydrophilic coating is one or more of the following: Plasma, polymer hydrophilic coating, nano-hydrophilic coating, or hydrophilic grafting.

7. A digital PCR microfluidic chip according to claim 5, characterized in that, The hydrophilic reagent is bonded to the inner or outer surface of the micropores by vapor deposition, vapor grafting, spraying, or impregnation.

8. A digital PCR microfluidic chip according to claim 1, characterized in that, The channel layer (2) is made of one or more of PC, COC, COP, PET, and double-sided adhesive.

9. A digital PCR microfluidic chip according to claim 1, characterized in that, The microporous layer (1) and the sealing layer (3) are made of one or more of PC, COC, COP and PET.

10. A digital PCR microfluidic chip according to claim 1, characterized in that, The microporous layer (1), channel layer (2), and sealing layer (3) are bonded together by adhesive bonding, hot-press bonding, or solvent bonding.

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