Centrifugal liquid drop generation micro-fluidic chip and digital PCR (polymerase chain reaction) detection method

Abstract

The invention relates to the technical field of microfluidics and digital PCR detection, and discloses a microfluidic chip for centrifugal droplet generation and a digital PCR detection method.The microfluidic chip comprises a sealed cavity formed by a bottom plate and a cover plate, a through hole is formed in the center of the chip and used for being fixed to centrifugal equipment, the core structure of the chip is a droplet generation component, and the droplet generation component is arranged in the cavity. Comprising a sample adding cavity, a micro-nozzle array and a liquid drop collecting cavity, a sample is driven by centrifugal force to generate nano-liter-scale'water-in-oil 'liquid drops through micro-nozzles, the chip is used for digital PCR reaction, each liquid drop serves as an independent reaction unit to improve detection sensitivity and precision, the chip is suitable for detection of low-abundance gene mutation, and by combining Taqman-MGB probe or other molecule detection technologies, the detection sensitivity and accuracy are improved. The chip can efficiently detect mutation types of EGFR genes, including EGFR-T790M mutation, has a wide clinical application prospect, provides a high-throughput and high-precision droplet generation method, and is suitable for application scenarios of digital PCR, single cell analysis and other microfluidic technologies.

Description

Technical Field

[0001] The present invention relates to the field of microfluidics technology and digital PCR detection technology, in particular to a microfluidics chip for centrifugal droplet generation and a digital PCR detection method. Background Art

[0002] Digital PCR (dPCR) is a new molecular biology technology based on polymerase chain reaction (PCR) for accurate quantitative detection of DNA or RNA molecules. dPCR achieves absolute quantification at the single-molecule level by distributing samples into tens to tens of thousands of independent reaction units, each of which undergoes independent PCR amplification.

[0003] The advantages of dPCR are:

[0004] 1. Absolute quantification, the result determination does not depend on the cycle threshold of amplification;

[0005] 2. Low sample requirement, which has obvious advantages in detecting precious or degraded samples;

[0006] 3. High sensitivity, which can accurately detect tiny differences in target fragments;

[0007] 4. High tolerance, reducing the interference of background sequences and inhibitors on the reaction;

[0008] Therefore, dPCR has good application prospects in the detection of low-abundance ctDNA with complex background.

[0009] According to the different sample separation methods, digital PCR is mainly divided into two categories: microarray chip type and droplet digital PCR. Microarray chip mainly has tens of thousands of square arrays of holes of the same volume etched on the chip, and nanoliters of liquid are sealed in a high-throughput micro-pool or micro-channel for subsequent PCR amplification and direct interpretation of the results after amplification. Droplet digital PCR is mainly designed with a chip with microfluidic channels and reaction chambers. Thousands of tiny droplets are obtained in the reaction chamber by separating the oil and water phases. This PCR reaction system with a single oil-in-water droplet as the unit is easier to achieve small volume and high throughput, and the system is simple and low cost.

[0010] Common droplet generation methods include the "T" channel method, the flow focusing method and the coaxial method. Driven by stable positive or negative pressure, these three droplet generation methods have good consistency (CV <5%) and generation rate (about 200-1000).

[0011] The number of droplets, generation rate and droplet consistency are key factors affecting the application of droplet microtechnology. In order to achieve higher throughput droplet generation, researchers used "T" channel method, flow focusing method and coaxial method for array and droplet preparation. In 2011, Jan et al. performed 16 arrays on the "T" droplet generation structure, which increased the droplet preparation rate by 16 times. In 2013, YANG, XU et al. performed 64 arrays on the flow focusing micro generation structure, which increased the generation rate by 64 times. In 2017, VENKATA et al. performed 200 arrays on the flow focusing droplet generation structure, which increased the generation rate by 200 times. Although this array structure increases the generation rate, due to the size and flow rate errors of each generated droplet structure, the generated droplet size varies greatly (CV>15%), which affects the equal distribution of samples and the final accuracy and sensitivity.

[0012] The other method is to use a multi-stage droplet segmentation method to gradually divide the generated droplets into small droplets to increase the generation rate. The basic principle is: the first stage divides the "T-type" or "+" type droplets into two, and the second stage divides the droplets divided in the first stage into two. With each additional stage, the number of droplets doubles. The rate of droplet generation is exponentially related to the number of stages. In 2012, KAWAI, FUJII et al.

[24] used a 5-stage segmentation method to increase the droplet generation rate by 25 times. In 2015, HATCH, FISHER et al.

[25] used an 8-stage segmentation method to increase the droplet generation rate by 28 times. However, there are processing differences among the micro-segmentation structures, which makes the droplet segmentation asymmetric and the prepared droplet sizes vary greatly (CV>20%).

[0013] The above two high-throughput droplet generation technologies are difficult to apply to fields such as digital PCR due to their large size differences. The control of microfluidics requires expensive and sophisticated equipment, such as micro-injection pumps, integrated pneumatic pumps, etc.

[0014] To this end, we propose a microfluidic chip for centrifugal droplet generation and a digital PCR detection method. Summary of the invention

[0015] In order to make up for the deficiencies of the prior art, the present invention provides the following technical solutions: a microfluidic chip for centrifugal droplet generation and a digital PCR detection method, comprising a chip including a bottom sheet and a cover sheet sealed with the bottom sheet, the bottom sheet and the cover sheet forming a sealed cavity;

[0016] A through hole is provided at the center of the chip, and a positioning notch is provided at the inner edge of the through hole for fixing the chip on the centrifugal device;

[0017] The chip comprises at least one independent droplet generation component, which comprises a sample addition chamber, a micro-nozzle array and a droplet collection chamber. The droplet generation component can generate droplets under the action of centrifugal force.

[0018] Preferably, the sample inlet is capable of accommodating 20 to 200 microliters of sample, and the continuous phase inlet is used to add fluorinated oil to support droplet generation.

[0019] Preferably, the droplet generation component includes a quantitative pool, which accurately distributes the sample through centrifugal force. The quantitative pool automatically distributes the sample volume through an overflow metering structure to ensure that the sample volume entering the droplet generation unit is 10.5 μL.

[0020] Preferably, the quantitative pool is provided with an exhaust hole to prevent gas from interfering with sample quantification and distribution.

[0021] Preferably, the micro-nozzle structure array includes 5 to 300 micro-nozzle structures, and the diameter of the micro-nozzle structure is 10 to 100 μm.

[0022] Preferably, the micro-nozzle structure array uses centrifugal force to push the sample through the micro-nozzle structure to generate "water-in-oil" droplets with a diameter of 10 to 100 μm, and the droplets enter the droplet collection chamber through the micro-nozzle structure.

[0023] Preferably, the droplet collection chamber is pre-stored with fluorinated oil as a continuous phase for droplet generation to prevent cross contamination between droplets.

[0024] Preferably, the serpentine microchannel allows the sample to be fully mixed and reach a stable flow before entering the sample storage tank. The sample in the storage tank enters the quantitative tank corresponding to the droplet generation unit through centrifugation, and the excess sample enters the waste liquid tank. The quantitative tank is provided with an exhaust hole. After the centrifugal speed changes, the sample in the quantitative tank enters the droplet generation unit through the microfluidic channel and the micronozzle structure. The droplet generation unit is filled with the continuous phase reagent through the oil inlet hole and the oil storage tank.

[0025] Preferably, the method comprises the following steps:

[0026] Step 1: Add the digital PCR reaction system to the sample inlet of the chip;

[0027] Step 2: The dispersed phase reagent and the continuous phase reagent are respectively transported to the quantitative pool and the droplet generation unit by centrifugal force to generate nanoliter-scale "water-in-oil" droplets; the generated droplets are used for PCR amplification, and each droplet serves as an independent PCR reaction unit;

[0028] Step 3: Use fluorescence detection technology to detect the amplified product to determine the target nucleic acid content in the sample.

[0029] Beneficial Effects

[0030] Compared with the prior art, the present invention provides a microfluidic chip for centrifugal droplet generation and a digital PCR detection method, which have the following beneficial effects:

[0031] 1. Modular setup, consisting of a drive unit and a rotary module, for rapid liquid processing.

[0032] 2. Good repeatability, the inertial momentum of the centrifugal drive can suppress the disturbance of high-frequency pressure.

[0033] 3. Low loss, can eliminate the problem of bubbles in the injection pump interface.

[0034] 4. High-throughput droplet preparation. The chip can be designed with multiple independent droplet generation components. Each droplet generation unit is designed with a micro-nozzle array. The micro-nozzle array is composed of several micro-nozzles. When generating droplets, all micro-nozzles can prepare droplets at the same time.

[0035] Each independent droplet generation component includes a sample addition chamber, a micronozzle array, a droplet collection chamber, etc. Oil is pre-stored in the droplet collection chamber. Driven by centrifugal force, the aqueous sample flows along the microchannel to the micronozzle array, forming "oil-in-water" droplets that enter the collection chamber, and further coupled with highly specific Taqman-MGB fluorescence quantitative PCR technology, ARMS-PCR (Allele-Specific Oligonucleotide-PCR) or BDA-PCR (Blocker displacement amplification-PCR) amplification technology, ultimately achieving high-throughput and quantitative detection of low-abundance nucleic acid fragments in biological samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is a schematic diagram of the overall structure of the microfluidic chip for centrifugal droplet generation and the digital PCR detection method of the present invention;

[0037] Figure 2 A schematic diagram of a microfluidic chip for centrifugal droplet generation and an independent droplet generation component of a microfluidic chip in a digital PCR detection method of the present invention;

[0038] Figure 3 Fluorescence microscope images of the microfluidic chip for centrifugal droplet generation and the microfluidic chip for digital PCR detection of EGFR wild-type reference substances in the detection method of the present invention;

[0039] Figure 4 Fluorescence microscope images of the microfluidic chip for centrifugal droplet generation and the microfluidic chip in the digital PCR detection method of the present invention detecting an EGFR-T790M reference substance (mutation rate 1:2000).

[0040] Figure 5 The microfluidic chip for centrifugal droplet generation and the fluorescence microscope image detected by the fluorescence spectrum diagram (mutation rate 1:200) of the fluorescent microspheres prepared by the microfluidic chip in the digital PCR detection method of the present invention.

[0042] In the figure: 1. Sample inlet; 2. Serpentine microchannel; 3. Oil inlet hole; 4. Liquid storage tank; 5. Exhaust hole; 6. Oil storage tank; 7. Quantification tank; 8. Waste liquid tank; 9. Droplet generation unit; 10. Micro nozzle structure. DETAILED DESCRIPTION

[0043] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0044] like Figure 1-2 As shown, the microfluidic chip bottom sheet and the cover sheet sealed with the bottom sheet are firmly attached with double-sided adhesive having the required bonding strength, being able to withstand high temperatures, and having no significant adverse effects on subsequent PCR reactions. A through hole is provided in the center of the microfluidic chip, and a positioning notch is provided at the inner edge of the through hole. The through hole and the positioning notch jointly play a role in fixing and positioning, and only one centrifuge is needed to complete the centrifugal distribution of the sample.

[0045] like Figure 1 As shown, a plurality of independent droplet generating components are provided on the bottom film, and a single droplet generating component is provided with a sample inlet 1 on the sample loading chamber. For example, when the required loading volume is between 20 and 200 microliters, the diameter of the sample inlet 1 can be between 0.5 mm and 2 mm.

[0046] like Figure 2 As shown, under the action of centrifugal force, the sample can pass through the serpentine microchannel 2 so that the sample can be fully mixed and achieve flow stability, so as to enter the sample storage tank 4. The sample in the storage tank 4 can enter the quantitative tank 7 corresponding to the droplet generation unit 9 through centrifugation, and the excess sample enters the waste liquid tank 8. The quantitative tank 7 is provided with an exhaust hole 5. As the centrifugal speed changes, the sample in the quantitative tank 7 enters the droplet generation unit 9 through the microchannel and micronozzle structure 10. The droplet generation unit 9 is filled with the continuous phase reagent through the oil inlet hole 3 and the oil storage tank 6.

[0047] The sample inlet 1 and the continuous phase inlet in the microfluidic chip are connected through an independent microfluidic channel to ensure the smooth input of the dispersed phase and the continuous phase. The quantitative pool 7 is the key part of droplet generation. The sample is accurately distributed by centrifugation. The quantitative pool 7 automatically distributes the sample through the overflow metering structure to ensure that the sample volume entering the droplet generation unit 9 is consistent, and the volume is 10.5 μL, which avoids manual intervention and improves the distribution accuracy.

[0048] The droplet generation unit 9 includes an array of 5 to 300 micro-nozzle structures 10, and the diameter of the micro-nozzle structure 10 is designed to be 10 to 100 μm. During the centrifugation process, the dispersed phase sample is squeezed into the droplet collection chamber of the pre-stored oil phase through the nozzle to form "oil-in-water" droplets. The array of micro-nozzle structures 10 can generate a large number of droplets at the same time, which greatly improves the efficiency and consistency of droplet generation. After the droplets are generated, under the action of centrifugal force, the droplets enter the droplet collection chamber from the micro-nozzle structure 10. Fluorinated oil is pre-stored in the collection chamber as a continuous phase to stabilize the generated droplets. By controlling the centrifugal speed, the droplets can be evenly distributed in the collection area to form a regular single-layer arrangement, which is convenient for subsequent reactions and detection.

[0049] Microfluidic chips can be mainly used in the field of digital PCR. Targeted therapy based on epidermal growth factor receptor (EGFR) gene mutation has gradually become a part of the comprehensive treatment of non-small cell lung cancer (NSCLC). However, long-term application of targeted therapy often causes acquired drug resistance, of which about 60% of acquired drug resistance is due to secondary EGFR-T790M mutation. Therefore, the present invention uses EGFR-T790M mutation as the detection target, optimizes and evaluates the high-throughput centrifugal droplet generation chip and digital PCR detection method provided by the present invention, and confirms the clinical practicality of the present invention. The specific steps are as follows:

[0050] Preparation of test samples: H1975 genomic DNA (carrying EGFR-T790M mutation) was used as a positive control, A549 genomic DNA (EGFR wild type) was used as a negative control, EGFR-T790M mutation and EGFR wild type were diluted and mixed according to different copy numbers, and reaction templates with mutation ratios (mutant / wild type) of 0:1, 1:2000, 1:200, and 1:10 were prepared, and the ddPCR reaction system was established and optimized;

[0051] Preparation of ddPCR reaction system: The digital PCR system includes the sample to be tested, 500nM PCR upstream and downstream primers, 200nM mutant Taqman-MGB probe, and 11uL of 2x digital PCR premix, including 4μL of positive control and 8μL of negative control. The reaction system is less than 22μL, which is made up with enzyme-free water;

[0052] Table 1 Sequences and dosages of primers and probes in the examples

[0053]

[0054] Sample loading and droplet generation: Add the prepared ddPCR reaction system to the sample inlet 1 of the chip, place the chip in a self-developed centrifugal droplet generator, and the sample is distributed into the quantitative pool 7 by centrifugal force, and then generates nanoliter droplets through the micro-nozzle structure 10. These droplets are wrapped in the oil phase to prevent cross contamination between droplets. The specific droplet generation centrifugation procedure is as follows:

[0055] Table 2 Droplet generation centrifugation program

[0056]

[0057] PCR reaction: The microfluidic chip with generated droplets is placed in a flat-plate in-situ PCR instrument for PCR amplification. The amplification program is set as follows: 95°C pre-denaturation for 10 min, 94°C denaturation for 30 s, 55-63°C annealing / extension for 120 s, and the temperature is lowered to 16°C after 40 cycles of denaturation, annealing and extension. Each droplet is treated as an independent reaction unit. Since the droplets are evenly distributed and have the same volume, the amplification results are highly consistent, which is convenient for subsequent fluorescence detection and quantitative analysis.

[0058] like Figure 3-5 As shown, fluorescence detection and data analysis: After PCR amplification is completed, the amplification results are detected by fluorescence microscopy, and the fluorescence channel is selected as "FAM". The nucleic acid amplification in each droplet is presented by fluorescence signals. The number of fluorescent droplets is proportional to the abundance of the target nucleic acid. When the detection template is EGFR wild type, no droplets with fluorescent signals appear under the fluorescence microscope. When the ratio of mutant type to wild type is 1:2000, droplets with fluorescent signals appear under the microscope to detect mutations. Therefore, the lower limit of mutations that can be actually detected by this method is 0.05% ( Figure 4 ), with the increase of the ratio of mutant to wild type, when the mutation abundance reached 1:200 and 1:10, the number of droplets with fluorescent signals continued to increase, indicating that the digital PCR method based on the microfluidic chip in the present invention has a good signal response to the mutation abundance of EGFR-T790M.

[0059] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0060] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

[0061] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A microfluidic chip for centrifugal droplet generation and a digital PCR detection method, characterized in that: The chip comprises a bottom sheet and a cover sheet sealed with the bottom sheet, wherein the bottom sheet and the cover sheet form a sealed cavity; A through hole is provided at the center of the chip, and a positioning notch is provided at the inner edge of the through hole for fixing the chip on the centrifugal device; The chip comprises at least one independent droplet generation component, which comprises a sample addition chamber, a micro-nozzle array and a droplet collection chamber. The droplet generation component can generate droplets under the action of centrifugal force.

2. The microfluidic chip for centrifugal droplet generation and the digital PCR detection method according to claim 1, characterized in that: The sample inlet (1) can accommodate 20 to 200 microliters of sample, and the continuous phase inlet is used to add fluorinated oil to support droplet generation.

3. The microfluidic chip and digital PCR detection method for centrifugal droplet generation according to claims 1-2, characterized in that: The droplet generation component comprises a quantitative pool (7), which accurately distributes the sample through centrifugal force. The quantitative pool (7) automatically distributes the sample volume through an overflow metering structure to ensure that the sample volume entering the droplet generation unit (9) is 10.5 μL.

4. The microfluidic chip for centrifugal droplet generation and the digital PCR detection method according to claim 3, characterized in that: The quantitative pool (7) is provided with an exhaust hole (5) to prevent gas from interfering with sample quantification and distribution.

5. The microfluidic chip and digital PCR detection method for centrifugal droplet generation according to claims 1-4, characterized in that: The micro-nozzle structure (10) array comprises 5 to 300 micro-nozzle structures (10), and the diameter of the micro-nozzle structure (10) is 10 to 100 μm.

6. The microfluidic chip for centrifugal droplet generation and the digital PCR detection method according to claim 5, characterized in that: The micro-nozzle structure (10) array uses centrifugal force to push the sample through the micro-nozzle structure (10), generating "water-in-oil" droplets with a diameter of 10 to 100 μm, and the droplets enter the droplet collection chamber through the micro-nozzle structure (10).

7. The microfluidic chip for centrifugal droplet generation and the digital PCR detection method according to claims 1-6, characterized in that: The droplet collection chamber is pre-stored with fluorinated oil as the continuous phase for droplet generation to prevent cross contamination between droplets.

8. The microfluidic chip for centrifugal droplet generation and the digital PCR detection method according to claim 1, characterized in that: The serpentine microchannel (2) allows the sample to be fully mixed and enter the sample liquid storage tank (4) after reaching a stable flow. The sample in the liquid storage tank (4) enters the quantitative tank (7) corresponding to the droplet generation unit (9) through centrifugation, and the excess sample enters the waste liquid tank (8). The quantitative tank (7) is provided with an exhaust hole (5). After the centrifugal speed changes, the sample in the quantitative tank (7) enters the droplet generation unit (9) through the microchannel and micronozzle structure (10), and the droplet generation unit (9) is filled with a continuous phase reagent through the oil inlet hole (3) and the oil storage tank (6).

9. The microfluidic chip for centrifugal droplet generation and the digital PCR detection method according to claims 1-7, characterized in that: The following steps are involved: Step 1: Add the digital PCR reaction system to the sample inlet of the chip (1); Step 2: The dispersed phase reagent and the continuous phase reagent are respectively transported to the quantitative pool (7) and the droplet generation unit (9) by centrifugal force to generate nanoliter-scale "water-in-oil" droplets; the generated droplets are used for PCR amplification, and each droplet serves as an independent PCR reaction unit; Step 3: Use fluorescence detection technology to detect the amplified product to determine the target nucleic acid content in the sample.

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