WO2023015758A1

WO2023015758A1 - System for implementing high-throughput integrated microdroplet digital pcr

Info

Publication number: WO2023015758A1
Application number: PCT/CN2021/130781
Authority: WO
Inventors: 夏焕明; 肖博
Original assignee: 北京慧智医疗器械有限公司
Priority date: 2021-08-11
Filing date: 2021-11-16
Publication date: 2023-02-16
Also published as: CN113604344B; CN113604344A

Abstract

Provided is a system for implementing high-throughput integrated microdroplet digital PCR, belonging to the technical field of nucleic acid detection and analysis. The system comprises a microdroplet preparation unit, a PCR amplification unit and a fluorescence detection and analysis unit; the microdroplet preparation unit comprises a driver, a disturbance device and a microdroplet generator; the microdroplet generator comprises an oil phase channel, a sample channel and a detection chamber; the driver generates a drive signal and acts upon the disturbance device; the disturbance device generates a velocity pulse in the oil phase, and then a mixed liquid of an oil-phase shear sample and a PCR reaction reagent, generating microdroplets; the microdroplets are spread uniformly within the detection chamber, and the PCR amplification unit performs a PCR amplification reaction on the microdroplets in the detection chamber; then, the fluorescence detection and analysis unit performs data reading and analysis on the microdroplets so as to complete quantitative detection of the specific nucleic acid. Achieved are a rapid preparation of microdroplets, reduced oil consumption, reduced microdroplet size dependence on the size of a fluid channel, high detection precision and reliability, and low cost.

Description

A high-throughput integrated micro-droplet digital PCR realization system

technical field

The invention relates to a high-throughput integrated micro-droplet digital PCR realization system, which belongs to the technical field of nucleic acid detection and analysis.

Background technique

In the field of life science research, PCR (polymerase chain reaction) is an extremely common nucleic acid detection and analysis method. At present, when performing nucleic acid detection, the mainstream droplet digital PCR instrument usually includes three parts: a droplet generation system, a thermal cycler, and a signal reading system. The phase (continuous phase) squeezes the sample water phase (discrete phase) to disperse the sample into several micro-droplets, then place the sample that has become micro-droplets in a sample tube, and place the sample tube in a thermal cycler for amplification. , and then put the sample tube in the signal reading system for detection. The detection principle of the traditional signal reading system is: micro-droplets pass through the channel quickly one by one, and the micro-droplets are detected by laser.

Traditional micro-droplets are mostly generated in a passive way, and the droplet size, frequency, and multiple parameters such as device channel size, flow rate, and physical properties are interrelated and difficult to adjust. Even with fixed operating parameters, the droplet size remains difficult to precisely control due to the batch-to-batch variation in chip size. Passive methods are used to prepare micro-droplets, and the size of the required fluid channel is relatively small, which requires high machining accuracy and increases the cost; if a larger channel size is used, the flow rate of the oil phase must be increased, and redundant fluid must be removed in the follow-up work. oil, the process is complex.

At present, in the research of preparing micro-droplets in an active way, most of the external disturbance frequency and droplet generation frequency are not in one-to-one correspondence, and the droplet generation frequency and size still need to be calibrated; and the droplet generation frequency is generally low (< 100Hz); the droplet size range is narrow.

In addition, the current micro-droplet digital PCR involves micro-droplet preparation, amplification reaction, and detection and analysis. At present, related products are completed with multiple equipment and multiple steps. Droplet generation, PCR, and detection are three different systems, and the operation is relatively complicated. There are many factors that affect the final result of PCR, each test takes a long time, and the cost of equipment is high. The tested samples need to be processed separately to prevent cross-contamination.

Contents of the invention

Aiming at the deficiencies in the prior art, the present invention provides a high-throughput integrated micro-droplet digital PCR implementation system, adopts active control technology to prepare micro-droplets, realizes rapid preparation of micro-droplets, and reduces oil consumption ( continuous phase), while reducing the dependence of the size of the micro-droplet on the size of the fluid channel, improving the uniformity of the size of the micro-droplet, high detection accuracy and reliability, and low cost.

The technical solution of the present invention to solve the above technical problems is as follows: a high-throughput integrated micro-droplet digital PCR realization system, the micro-droplet digital PCR realization system includes a micro-droplet preparation unit, a PCR amplification unit and a fluorescence detection analysis unit;

The micro-droplet preparation unit includes a driver, a disturbance device, and a micro-droplet generator. The micro-droplet generator includes an oil phase channel, a sample channel, and a detection chamber. The oil phase channel is connected with an oil phase. There is a discrete phase in the sample channel, and the discrete phase is a mixed liquid of the sample to be detected and the PCR reaction reagent, and the oil phase channel and the sample channel are connected to the detection chamber after converging;

The power supply is connected to the driver, and the driver can realize the amplification of the electric signal. The amplified driving signal generated by the driver acts on the disturbance device, and the disturbance device converts the electric signal into a vibration signal. The disturbance device acts on the oil phase, and the disturbance device acts on the oil phase. The velocity pulse is generated in the phase, and then the oil phase shears the discrete phase in the micro-droplet generator to generate micro-droplets; the generated micro-droplets are collected in the detection chamber, and the micro-droplets are evenly spread in the detection chamber, and PCR The amplification unit heats the micro-droplets in the detection chamber to realize the PCR amplification reaction; then the fluorescence detection and analysis unit directly reads and analyzes the data of the micro-droplets after the PCR amplification reaction, and completes the nucleic acid quantitative detection of the sample . Since the speed pulse corresponds to the generation of micro-droplets, that is, one pulse generates one micro-droplet, the frequency of generating micro-droplets is consistent with the frequency of the speed pulse and the frequency of the driving signal. For convenience, the discrete phase mentioned in this description refers to the mixed liquid containing the sample and the reagents required for the PCR reaction, that is, the micro-droplets in the description of the present invention are all mixed liquids containing the sample and the reagents required for the PCR reaction. Micro-droplets.

The beneficial effects of the present invention are:

(1) The present invention applies external disturbances to the continuous phase (oil phase), and through the generation of velocity pulses to enhance the shearing effect, the generation of micro-droplets is actively controlled, and the droplet size and frequency are not affected by the oil phase channel when the droplets are passively generated. Difficult to adjust and calibrate due to the influence of multiple parameters such as size, sample channel size, two-phase flow rate and flow rate ratio;

(2) The present invention generates micro-droplets controlled by flow rate and electrical signal, and directly controls the droplet size through the discrete phase flow rate, thereby avoiding the error caused by the work difference during mass production of chips to the generation of micro-droplets, reducing the Hardware production precision requirements;

(3) The size of the micro-droplet is not affected by the size of the chip channel within the working range of the technology of the present invention, and can be directly adjusted by the discrete phase flow rate: V=Q _d /f, V is the droplet volume, Q _d is the discrete phase flow rate, f is the disturbance frequency of the disturbance device. The uniformity of micro-droplets is only affected by the flow stability, and the uniformity is good. The generation frequency of micro-droplets is synchronized with the disturbance frequency, which is convenient for adjusting the droplet size;

(4) The technology of the present invention can realize high-frequency and high-amplitude disturbance, and can realize the generation of micro-droplets at a rate of more than one hundred to several thousand or even higher per second. The micro-droplet preparation efficiency is high, and the micro-droplet is generated continuously and at high throughput. Compared with the passive method, the oil consumption (continuous phase) or oil-water ratio is greatly reduced under the condition of large channel size and high generation frequency, which is beneficial to the subsequent on-line Processing and detection can make there are tens of thousands to millions of micro-droplets in the detection chamber, ensuring that there are more micro-droplets in the detection chamber than in the prior art to obtain more accurate statistical data; and all The continuous phase flow rate needs to be low, which reduces the difficulty of subsequent collection and treatment of droplet products;

(5) The micro-droplet digital PCR realization system of the present invention has simple structure, low equipment cost, no need to use sample tubes, can realize the integrated design of micro-droplet preparation, PCR amplification and fluorescence detection and analysis, and is easy to operate, and the equipment low cost;

(6) The micro-droplet digital PCR implementation system of the present invention can be matched with a trace cell enrichment and sorting module to realize high-sensitivity detection of trace cells and trace nucleic acids.

On the basis of above-mentioned technical scheme, the present invention can also be improved as follows:

Further, the oil phase channel, the sample channel and the detection chamber are arranged on the droplet chip, the oil phase and the sample enter the oil phase channel and the sample channel respectively through the delivery pump, and the disturbance device acts on the oil phase generation speed pulse, and then the oil phase that generates the speed pulse converges with the sample in the sample channel, and the oil phase shears the sample into micro-droplets, and the micro-droplets enter the detection chamber, and the detection chamber is provided with an outlet; The cyclic heating mechanism cyclically heats the droplet chip to realize PCR amplification reaction; the fluorescence detection and analysis unit includes a high-sensitivity camera, and the high-sensitivity camera takes pictures of the micro-droplets of the droplet chip. The relative positions of the circulation heating mechanism, the high-sensitivity camera and the droplet chip are set according to design requirements.

The beneficial effect of adopting the above further scheme is: by applying external pulse disturbance to the oil phase, the shearing effect is strengthened, and the generation of micro-droplets is actively controlled; due to the small amount of oil used, the collection and detection of micro-droplet samples are directly integrated in the liquid On the drop chip, the used drop chip is treated as a disposable consumable together with the waste liquid to avoid cross-contamination between samples, and it is convenient to dispose of medical waste;

A high-sensitivity camera is used to take pictures at one time to read and analyze the data of micro-droplets after PCR amplification reaction. Compared with the traditional laser detection method, the efficiency is higher and the accuracy is higher; The droplet chip is cyclically heated to realize the PCR amplification reaction, and the high-sensitivity camera is used to take pictures of the micro-droplets for data reading. Compared with the current traditional mainstream micro-droplet digital PCR instrument, the structure can be integrated, so that the structure is more compact. It is more convenient to use.

Further, the disturbance device is a piezoelectric sheet, a piezoelectric ceramic tube or an eccentric wheel vibrator, and the delivery pump is a syringe pump or a pressure pump, preferably a pressure pump, but the delivery pump is not limited to only selecting a syringe pump or a pressure pump. Pump.

The beneficial effect of adopting the above-mentioned further scheme is that the piezoelectric sheet, piezoelectric ceramic tube or eccentric wheel vibrator is used as the disturbance device, the amplitude is relatively large, the frequency is high, and a large fluid channel can be used to generate high-flux micro-droplets. Moreover, the cost is low; using a pressure pump for fluid drive is convenient for users to operate, and only needs to calibrate the flow rate in the early stage, and then solidify the operating parameters.

Further, the disturbance device and the droplet chip are installed independently, and the oil passage at the outlet of the delivery pump that transports the oil phase first passes through the disturbance device and then connects to one end of the communication pipeline, and the other end of the communication pipeline is connected to the oil phase channel, the disturbing device is a piezoelectric sheet, and one or several piezoelectric sheets are installed on the wall of the oil passage, or piezoelectric sheets are installed on the upper and lower walls of the oil passage.

The beneficial effect of adopting the above-mentioned further solution is that the disturbance device is externally installed as an independent component, the droplet chip is a disposable consumable, and the disturbance device can be reused for a certain number of times before being replaced, reducing the cost of use;

Affected by the response time of the piezoelectric film, the disturbance intensity usually decays rapidly as the frequency increases, so the driving frequency of the piezoelectric film should be close to its resonance frequency to maintain a large amplitude at high frequencies. When the disturbance device is installed externally, the channel connecting the downstream of the oil phase to the micro-droplet generator increases, and the amplitude attenuates. However, simply increasing the area of the piezoelectric sheet will cause a decrease in the resonance frequency and affect the droplet preparation efficiency. Multiple piezoelectric sheets are embedded on the wall of the oil passage, which can be driven synchronously to maintain high-frequency and high-amplitude disturbances to the oil phase, and the cost of the piezoelectric sheets is very low.

Further, the oil phase channel and the sample channel have a T-shaped cross-flow structure, a Y-shaped cross-flow structure, a cross-shaped flow focusing structure or a co-flow structure on the droplet chip, and the co-flow structure is an oil phase channel socket inside the sample channel.

The beneficial effect of adopting the above further scheme is: the oil phase channel and the sample channel adopt a T-shaped cross-flow structure, a Y-shaped cross-flow structure, a cross-shaped flow focusing structure or a co-flow structure, all of which can realize the shearing of the oil relative to the discrete phase, and realize Preparation of microdroplets.

Further, several groups of micro-droplet generators are provided on the droplet chip, and each group of micro-droplet generators includes an oil phase channel, a sample channel and a detection chamber, and a single disturbance device is used to drive all oil circuits or each Oil circuits are equipped with independent disturbance devices.

The beneficial effect of adopting the above-mentioned further scheme is: several groups of micro-droplet generators are set on the droplet chip to realize multi-channel parallel operation, and one droplet chip can test multiple samples at the same time without causing cross-contamination; Sufficient cases can use a single disturbance device to drive multiple oil circuits simultaneously.

Further, a steering valve is provided at the outlet of each oil circuit, and the steering valve connects the oil circuit, the air pump and the oil phase channel.

The beneficial effect of adopting the above-mentioned further scheme is that when the multi-channels are running in parallel, the switching and cleaning between each oil circuit is realized by means of the steering valve. When the steering valve controls the air pump, the oil circuit and the oil phase channel are all disconnected, the oil phase The channel is not working, this function is used for switching between discrete phases of different samples; when the steering valve controls the air pump to disconnect from the oil phase channel, and the oil circuit is connected to the oil phase channel, the discrete phase enters the sample channel to form micro-droplets; The steering valve controls the disconnection of the oil circuit and the oil phase channel, and the connection between the air pump and the oil phase channel. The air pump inputs air into the oil phase channel, and blows a small amount of oil remaining in the outlet of the oil circuit and the oil phase channel into the detection chamber for cleaning. , to avoid the pollution of the working environment caused by the leakage of residual oil when the droplet chip is replaced.

Further, a blocking mechanism is provided at the entrance of the detection chamber, and a blocking mechanism is provided at the exit of the detection chamber.

The beneficial effect of adopting the above-mentioned further scheme is: before the PCR amplification reaction after the micro-droplets are generated, the blocking mechanism will seal the inlet and outlet of the detection chamber, so as to avoid the micro-droplets in the detection chamber from being damaged during the PCR amplification reaction. , the micro-droplet leaks from the detection chamber and affects the detection and analysis results; in addition, sealing the micro-droplet in the detection chamber can also avoid cross-contamination and environmental pollution between samples. The sealing mechanism can use an ultrasonic welding device, but It is not limited to use only ultrasonic welding devices.

Further, the driving signal generated by the driver is a triangular wave, a rectangular wave or a sine wave. Preferably, the driving signal generated by the driver is a rectangular wave; the generation frequency of the micro-droplets is consistent with the frequency of the driving signal generated by the driver; the generation frequency of the micro-droplets is greater than 100 per second.

The beneficial effect of adopting the above-mentioned further solution is that the staff can select the corresponding driving signal according to the requirements for the formation of micro-droplets and the performance of the disturbance device, and can adjust the waveform, frequency and amplitude as required.

Further, the realization system of micro-droplet digital PCR includes a GUI and a data analysis module, and the GUI and data analysis module control the operation of the realization system of micro-droplet digital PCR.

The beneficial effect of adopting the above-mentioned further scheme is that the GUI and the data analysis module can realize the related functions of the droplet chip placement module, pulse excitation control module, sample flow control module, detection chamber blocking module, temperature control module and fluorescence signal acquisition module. Hardware for control and data analysis.

Description of drawings

Fig. 1 is the schematic diagram of the working principle of the realization system of micro-droplet digital PCR in the embodiment 1-4;

2 is a top view and a front view of the droplet chip described in Example 1;

Fig. 3 is the typical temperature curve figure of cyclic heating among the embodiment 1-6;

Fig. 4 is a schematic diagram of the sealing mechanism sealing the inlet and outlet of the detection chamber in Embodiment 1-6

Fig. 5 is the schematic diagram of amplification reaction and fluorescent signal analysis in embodiment 1-6;

Fig. 6 is the structural schematic diagram of the fluorescence detection analysis unit in embodiment 1-6;

Fig. 7 is the view under different filters in the fluorescence detection process in embodiment 1-6;

8 is a top view of the perturbation device and droplet chip described in Example 2;

9 is a top view of the perturbation device and droplet chip described in Example 3;

Fig. 10 is a top view and an internal cross-sectional view of the installation of the oil circuit and the piezoelectric sheet described in Embodiment 4;

Fig. 11 is the working principle schematic diagram of embodiment 5;

12 is a schematic structural view of the droplet chip described in Embodiment 6;

Fig. 13 is the working principle schematic diagram of embodiment 6;

Fig. 14 is the working principle diagram of the steering valve in embodiment 6;

Fig. 15 is the micro-droplet detection figure obtained in embodiment 1;

Fig. 16 is the detection figure of the micro-droplets of different sizes obtained by controlling the flow rate of the discrete phase in Example 1;

In the figure, 1 oil phase channel, 2 sample channel, 3 outlet, 4 piezoelectric sheet, 5 detection chamber, 6 circulation heating mechanism, 7 oil circuit, 8 droplet chip, 9 connecting pipeline, 10 piezoelectric tube.

Detailed ways

In order to make the above objects, features and advantages of the present invention more obvious and comprehensible, specific implementations of the present invention will be described in detail below. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. However, the present invention can be implemented in many other ways different from those described here, and those skilled in the art can make similar improvements without departing from the connotation of the present invention, so the present invention is not limited by the specific embodiments disclosed below.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field of the invention. The terminology used herein in the description of the present invention is only for the purpose of describing specific embodiments, and is not intended to limit the present invention.

Example 1

A high-throughput integrated micro-droplet digital PCR realization system, including a micro-droplet preparation unit, a PCR amplification unit, a fluorescence detection and analysis unit, a blocking mechanism, a GUI and a data analysis module; the GUI and the data analysis module control the described Realization of micro-droplet digital PCR system hardware operation and data analysis;

The micro-droplet preparation unit includes a driver, a disturbance device and a micro-droplet generator, the perturbation device is a piezoelectric sheet 4, and the micro-droplet generator includes an oil phase channel 1, a sample channel 2 and a detection chamber 5 , the oil phase channel 1 is provided with an oil phase, and the sample channel 2 is provided with a discrete phase, the discrete phase is a mixed liquid of the sample to be detected and the PCR reagent, the oil phase channel 1 and the sample channel 2 After converging, it communicates with the detection chamber 5, and the detection chamber 5 is provided with an outlet 3; the oil phase channel 1, the sample channel 2 and the detection chamber 5 are arranged on the droplet chip 8, and the oil phase channel 1 and The sample channel 2 has a T-shaped cross-flow structure on the droplet chip 8, and the oil phase and the discrete phase enter the oil phase channel 1 and the sample channel 2 respectively through the delivery pump, and the delivery pump is a pressure pump;

The power supply is connected to the driver, the driver generates a driving signal and acts on the piezoelectric sheet 4, the piezoelectric sheet 4 acts on the oil phase, the piezoelectric sheet 4 generates speed pulses in the oil phase, and then generates the oil phase of the speed pulses Converging with the discrete phase in the sample channel 2, after the oil phase cuts the discrete phase into micro-droplets, the micro-droplets enter the detection chamber 5, and the micro-droplets are evenly spread in the detection chamber 5, the work flow chart As shown in FIG. 1 , the structure of the droplet chip 8 is shown in FIG. 2 .

The PCR amplification unit includes a circulation heating mechanism 6, the circulation heating mechanism 6 is located below the droplet chip 8, and the circulation heating mechanism 6 heats the micro-droplets in the detection chamber 5 to realize the PCR amplification reaction , the typical temperature curve of cyclic heating is shown in Figure 3, after the micro-droplets are generated and before the PCR amplification reaction, the sealing mechanism seals the inlet and outlet 3 of the detection chamber 5, and the sealing position is as shown in Figure 4; The fluorescence detection and analysis unit includes a high-sensitivity camera, which is located above the droplet chip 8. The high-sensitivity camera takes a one-time photo of the micro-droplets in the detection chamber 5, analyzes the biological indicators according to the fluorescence signal, and analyzes the biological indicators of the PCR amplification. After the reaction, the micro-droplets are read and analyzed to complete the nucleic acid quantitative detection of the sample. The process of fluorescence signal analysis is as follows: after the amplification is completed, the fluorescent signals in the detection chamber 5 are collected; the micro-droplets with fluorescent signals The drop is marked as 1, and the droplet without fluorescent signal is marked as 0; according to the total number of digital PCR reaction units (n), the number of units with fluorescent signal (f) and the dilution factor (m) of the sample, the sample’s Initial copy number (concentration c)

The principle of amplification reaction and fluorescence signal analysis is shown in FIG. 5 . In addition, the fluorescence detection and analysis unit can select an appropriate filter (filter) during the fluorescence signal collection process. The structure and principle of the fluorescence detection and analysis unit is shown in Figure 6, and the views under different filters are shown in Figure 7.

By controlling the flow conditions of the discrete phase (sample) and the continuous phase (oil phase), the size of the micro-droplets obtained is shown in Figure 15. Under the same driving frequency and the same channel conditions, micro-droplets of different sizes can be obtained by controlling the flow rate of the discrete phase, as shown in Figure 16. It can be seen from this that the present invention generates micro-droplets controlled by flow rate and electrical signal, and directly controls the size of the droplet through the discrete phase flow rate, thereby avoiding the work difference when mass-producing the droplet chip 8 to the micro-droplets. The error caused by generation reduces the precision requirement of hardware production.

Example 2

The micro-droplet preparation unit includes a driver, a disturbance device and a micro-droplet generator, the perturbation device is a piezoelectric sheet 4, and the micro-droplet generator includes an oil phase channel 1, a sample channel 2 and a detection chamber 5 , there is an oil phase in the oil phase channel 1, and a discrete phase in the sample channel 2, and the discrete phase is a mixed liquid of the sample to be detected and the PCR reagent, and the oil phase and the discrete phase are transported by a delivery pump , the oil phase channel 1 and the sample channel 2 are connected to the detection chamber 5 after converging, and the detection chamber 5 is provided with an outlet 3; the oil phase channel 1, the sample channel 2 and the detection chamber 5 are arranged on the On the chip 8, the oil phase channel 1 and the sample channel 2 have a T-shaped cross-flow structure on the droplet chip 8, the piezoelectric sheet 4 and the droplet chip 8 are installed independently, and the piezoelectric sheet 4 is externally placed. The outlet oil passage 7 of the delivery pump for delivering the oil phase first passes through the piezoelectric plate 4 and then connects to one end of the communication pipeline 9, the other end of the communication pipeline 9 is connected to the oil phase channel 1, and the delivery pump is a pressure pump. As shown in FIG. 8 , the piezoelectric sheet 4 is externally installed as an independent component, and the droplet chip 8 is a disposable consumable. The piezoelectric sheet 4 can be replaced after being reused for a certain number of times, thereby reducing costs.

The power supply is connected to the driver, the driver generates a driving signal and acts on the piezoelectric sheet 4, the piezoelectric sheet 4 acts on the oil phase, the piezoelectric sheet 4 generates speed pulses in the oil phase, and then generates the oil phase of the speed pulse Converge with the discrete phase in the sample channel 2, after the oil phase cuts the discrete phase into micro-droplets, the micro-droplets enter the detection chamber 5, and the micro-droplets are evenly spread in the detection chamber 5, the working principle flow The picture is shown in Figure 1.

The PCR amplification unit includes a circulation heating mechanism 6, which is located below the droplet chip 8, and the circulation heating mechanism 6 heats the micro-droplets in the detection chamber 5 to realize the PCR amplification reaction , the typical temperature curve of cyclic heating is shown in Figure 3, after the micro-droplets are generated and before the PCR amplification reaction, the sealing mechanism seals the inlet and outlet 3 of the detection chamber 5, and the sealing position is shown in Figure 4; The fluorescence detection and analysis unit includes a high-sensitivity camera, the high-sensitivity camera is located above the droplet chip 8, and the high-sensitivity camera takes a one-time photo of the micro-droplets in the detection chamber 5, analyzes the biological indicators according to the fluorescent signal, and performs PCR amplification. After the reaction, the micro-droplets are read and analyzed to complete the nucleic acid quantitative detection of the sample. The process of fluorescence signal analysis is as follows: after the amplification is completed, the fluorescent signal in the detection chamber 5 is collected; the micro-droplet with the fluorescent signal The drop is marked as 1, and the droplet without fluorescent signal is marked as 0; according to the total number of digital PCR reaction units (n), the number of units with fluorescent signal (f) and the dilution factor (m) of the sample, the sample’s The principles of initial copy number (concentration c), amplification reaction and fluorescence signal analysis are shown in FIG. 5 . In addition, the fluorescence detection and analysis unit can select a suitable filter (filter) during the fluorescence signal collection process. The structure principle of the fluorescence detection and analysis unit is shown in Figure 6, and the views under different filters are shown in Figure 7.

Example 3

The micro-droplet preparation unit includes a driver, a disturbance device and a micro-droplet generator, the disturbance device is a piezoelectric tube 10, and the structure of the piezoelectric tube 10 is simpler, and the micro-droplet generator includes an oil phase channel 1 , a sample channel 2 and a detection chamber 5, the oil phase channel 1 is provided with an oil phase, and the sample channel 2 is provided with a discrete phase, and the discrete phase is a mixed liquid of a sample to be detected and a PCR reaction reagent, the oil The phase and the discrete phase are transported by the delivery pump, and the oil phase channel 1 and the sample channel 2 are connected to the detection chamber 5 after converging, and the detection chamber 5 is provided with an outlet 3; the oil phase channel 1 and the sample channel 2 and the detection chamber 5 are arranged on the droplet chip 8, the oil phase channel 1 and the sample channel 2 have a T-shaped cross-flow structure on the droplet chip 8, and the piezoelectric tube 10 and the droplet chip 8 are installed independently Setting, the piezoelectric tube 10 is externally installed, and the outlet oil circuit 7 of the delivery pump that transports the oil phase first passes through the piezoelectric tube 10 and then connects to the oil phase channel 1. The delivery pump is a pressure pump, as shown in Figure 9, the piezoelectric The structure of the tube 10 is simpler, and the piezoelectric tube 10 is externally used as an independent component. The droplet chip 8 is a disposable consumable. The piezoelectric tube 10 can be reused after a certain number of times and replaced to reduce costs.

The power supply is connected to the driver, the driver generates a driving signal and acts on the piezoelectric tube 10, the piezoelectric tube 10 acts on the oil phase, the piezoelectric tube 10 generates a speed pulse in the oil phase, and then the oil phase of the speed pulse Converge with the discrete phase in the sample channel 2, after the oil phase cuts the discrete phase into micro-droplets, the micro-droplets enter the detection chamber 5, and the micro-droplets are evenly spread in the detection chamber 5, the working principle flow The picture is shown in Figure 1.

The PCR amplification unit includes a circulation heating mechanism 6, which is located below the droplet chip 8, and the circulation heating mechanism 6 heats the micro-droplets in the detection chamber 5 to realize the PCR amplification reaction , the typical temperature curve of cyclic heating is shown in Figure 3, after the micro-droplets are generated and before the PCR amplification reaction, the sealing mechanism seals the inlet and outlet 3 of the detection chamber 5, and the sealing position is shown in Figure 4; The fluorescence detection and analysis unit includes a high-sensitivity camera, the high-sensitivity camera is located above the droplet chip 8, and the high-sensitivity camera takes a one-time photo of the micro-droplets in the detection chamber 5, analyzes the biological indicators according to the fluorescent signal, and performs PCR amplification. After the reaction, the micro-droplets are read and analyzed to complete the nucleic acid quantitative detection of the sample. The process of fluorescence signal analysis is as follows: after the amplification is completed, the fluorescent signal in the detection chamber 5 is collected; the micro-droplet with the fluorescent signal The drop is marked as 1, and the droplet without fluorescent signal is marked as 0; according to the total number of digital PCR reaction units (n), the number of units with fluorescent signal (f) and the dilution factor (m) of the sample, the sample’s The principles of initial copy number (concentration c), amplification reaction and fluorescence signal analysis are shown in FIG. 5 . In addition, during the fluorescence signal acquisition process, the fluorescence detection and analysis unit can select a suitable filter. The structure principle of the fluorescence detection and analysis unit is shown in Figure 6, and the views under different filters are shown in Figure 7.

Example 4

The micro-droplet preparation unit includes a driver, a disturbance device and a micro-droplet generator, the perturbation device is a piezoelectric sheet 4, and the micro-droplet generator includes an oil phase channel 1, a sample channel 2 and a detection chamber 5 , there is an oil phase in the oil phase channel 1, and a discrete phase in the sample channel 2, and the discrete phase is a mixed liquid of the sample to be detected and the PCR reagent, and the oil phase and the discrete phase are transported by a delivery pump , the oil phase channel 1 and the sample channel 2 are connected to the detection chamber 5 after converging, and the detection chamber 5 is provided with an outlet 3; the oil phase channel 1, the sample channel 2 and the detection chamber 5 are arranged on the droplet On the chip 8, the oil phase channel 1 and the sample channel 2 have a T-shaped cross-flow structure on the droplet chip 8, the piezoelectric sheet 4 and the droplet chip 8 are installed independently, and the piezoelectric sheet 4 is externally placed. The outlet oil passage 7 of the delivery pump for delivering the oil phase first passes through the piezoelectric plate 4 and then connects to one end of the communication pipeline 9, the other end of the communication pipeline 9 is connected to the oil phase channel 1, and the delivery pump is a pressure pump. Several small piezoelectric sheets 4 are installed on the wall surface of the oil passage 7 , and small piezoelectric sheets 4 are provided on the upper and lower walls of the oil passage 7 . When the piezoelectric sheet 4 is placed externally, the channel connecting the downstream of the oil phase to the micro-droplet generator increases, and the amplitude attenuates. However, simply increasing the area of the piezoelectric sheet 4 will cause a decrease in the resonance frequency and affect the droplet preparation efficiency. A plurality of small piezoelectric sheets 4 are embedded in the wall, which can be driven synchronously to maintain high-frequency and high-amplitude disturbances to the oil phase. The installation diagram of the piezoelectric sheets 4 on the oil circuit 7 is shown in Figure 10 .

Example 5

The micro-droplet preparation unit includes a driver, a disturbance device, and a micro-droplet generator, and the micro-droplet generator includes an oil phase channel 1, a sample channel 2, and a detection chamber 5, and the oil phase channel 1 has a An oil phase, the sample channel 2 is connected with a discrete phase, the discrete phase is a mixed liquid of the sample to be detected and the PCR reaction reagent, the oil phase channel 1 and the sample channel 2 are connected to the detection chamber 5 after converging , the detection chamber 5 is provided with an outlet 3; the oil phase channel 1, the sample channel 2 and the detection chamber 5 are arranged on the droplet chip 8, and the oil phase channel 1 and the sample channel 2 are formed on the droplet chip 8 T-shaped cross-flow structure. The oil phase and the discrete phase enter the oil phase channel 1 and the sample channel 2 respectively through the delivery pump, and the delivery pump is a pressure pump; when multiple samples are detected at the same time, multiple droplet chips 8 are connected in parallel, and the working principle is shown in Figure 11 shown.

The power supply is connected to the driver, the driver generates a driving signal and acts on the disturbance device, each of the disturbance devices acts on the corresponding oil phase, the disturbance device generates a speed pulse in the oil phase, and then the oil phase and the sample of the speed pulse are generated The discrete phases in the channel 2 converge, and after the oil phase shears the discrete phases into micro-droplets, the micro-droplets enter the detection chamber 5, and the micro-droplets are evenly spread in the detection chamber 5.

Example 6

The micro-droplet preparation unit includes a driver, a disturbance device, and a micro-droplet generator, and the micro-droplet generator includes an oil phase channel 1, a sample channel 2, and a detection chamber 5, and the oil phase channel 1 has a An oil phase, the sample channel 2 is connected with a discrete phase, the discrete phase is a mixed liquid of the sample to be detected and the PCR reaction reagent, the oil phase channel 1 and the sample channel 2 are connected to the detection chamber 5 after converging , the detection chamber 5 is provided with an outlet 3; the oil phase channel 1, the sample channel 2 and the detection chamber 5 are arranged on the droplet chip 8, and the oil phase channel 1 and the sample channel 2 are formed on the droplet chip 8 T-shaped cross-flow structure, the droplet chip 8 is provided with several groups of micro-droplet generators, each group of micro-droplet generators includes an oil phase channel 1, a sample channel 2 and a detection chamber 5, realizing multi-channel parallel connection In operation, one droplet chip 8 can test multiple samples at the same time without causing cross-contamination. The oil phase and the discrete phase respectively enter the oil phase channel 1 and the sample channel 2 through the delivery pump, and the delivery pump is a pressure pump; the structure of the droplet chip 8 is shown in Figure 12, and its working principle is shown in Figure 13. There is a steering valve at the outlet of each oil circuit 7, and the steering valve is connected to the oil circuit 7, the air pump and the oil phase channel 1. The working state of the steering valve is shown in Figure 14, and the multi-channel parallel operation is realized by means of the steering valve. The conversion and cleaning between each channel, as shown in Figure 14, state (a): the air and oil circuit 7 are all disconnected from the downstream channel, which is convenient for the conversion between different samples; state (b): the air and the downstream channel are disconnected Open, the oil channel 7 is connected, the sample enters, and micro-droplets are formed; state (c): the oil channel 7 and the downstream channel are disconnected, the air channel is connected, the air pump inputs air into the oil phase channel 1, and the oil channel 7 exits A small amount of oil remaining in the oil phase channel 1 is blown into the detection chamber 5 to play a cleaning role, so as to avoid the leakage of residual oil when the droplet chip 8 is replaced and cause pollution to the working environment.

The power supply is connected to the driver, the driver generates a driving signal and acts on the disturbance device, the disturbance device acts on the oil phase, a single disturbance device is used to drive multiple oil circuits 7 at the same time, the disturbance device generates speed pulses in the oil phase, and then The oil phase that generates the velocity pulse converges with the discrete phase in the sample channel 2, and after the oil phase shears the discrete phase into micro-droplets, the micro-droplets enter the detection chamber 5, and the micro-droplets are evenly distributed in the detection chamber 5. spread out.

The technical features of the above-mentioned embodiments can be combined arbitrarily. To make the description concise, all possible combinations of the technical features in the above-mentioned embodiments are not described. However, as long as there is no contradiction in the combination of these technical features, should be considered as within the scope of this specification.

The above-mentioned embodiments only express several implementation modes of the present invention, and the descriptions thereof are relatively specific and detailed, but should not be construed as limiting the patent scope of the invention. It should be pointed out that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent for the present invention should be based on the appended claims.

Claims

A high-throughput integrated micro-droplet digital PCR realization system is characterized in that the micro-droplet digital PCR realization system includes a micro-droplet preparation unit, a PCR amplification unit, and a fluorescence detection and analysis unit;

The micro-droplet preparation unit includes a driver, a disturbance device, and a micro-droplet generator. The micro-droplet generator includes an oil phase channel, a sample channel, and a detection chamber. The oil phase channel is connected with an oil phase. There is a discrete phase in the sample channel, and the discrete phase is a mixed liquid of the sample to be detected and the PCR reaction reagent, and the oil phase channel and the sample channel are connected to the detection chamber after converging;

The power supply is connected to the driver, the driver generates a driving signal and acts on the disturbance device, the disturbance device acts on the oil phase, the disturbance device generates a velocity pulse in the oil phase, and then the oil phase is sheared and dispersed in the droplet generator The generated micro-droplets are collected in the detection chamber, and the micro-droplets are evenly spread in the detection chamber, and the PCR amplification unit heats the micro-droplets in the detection chamber to realize the PCR amplification reaction Then the fluorescence detection and analysis unit directly reads and analyzes the data of the micro-droplets after the PCR amplification reaction, and completes the quantitative detection of the nucleic acid of the sample.
A system for realizing high-throughput integrated micro-droplet digital PCR according to claim 1, wherein the oil phase channel, the sample channel and the detection chamber are arranged on the droplet chip, and the oil phase and the discrete phase The delivery pump enters the oil phase channel and the sample channel respectively, the disturbance device acts on the oil phase to generate a velocity pulse, and then the oil phase that generates the velocity pulse converges with the discrete phase in the sample channel, and the oil phase shears the discrete phase into After the micro-droplet, the micro-droplet enters the detection chamber, and the detection chamber is provided with an outlet; the PCR amplification unit includes a circulation heating mechanism, and the circulation heating mechanism performs circulation heating on the droplet chip Realize the PCR amplification reaction; the fluorescence detection and analysis unit includes a high-sensitivity camera, and the high-sensitivity camera takes pictures of the micro-droplets in the droplet chip.
A system for realizing high-throughput integrated micro-droplet digital PCR according to claim 2, wherein the disturbance device is a piezoelectric sheet, a piezoelectric ceramic tube or an eccentric wheel vibrator, and the conveying The pump is a syringe pump or a pressure pump.
A high-throughput integrated micro-droplet digital PCR implementation system according to claim 2, characterized in that the disturbance device and the droplet chip are installed independently, and the oil passage at the outlet of the delivery pump that transports the oil phase passes through first The disturbance device is connected to one end of the communication pipeline, and the other end of the communication pipeline is connected to the oil phase passage; the disturbance device is a piezoelectric sheet, and one or several piezoelectric sheets are installed on the wall of the oil passage, Or the upper and lower walls of the oil passage are provided with piezoelectric sheets.
The realization system of a high-throughput integrated micro-droplet digital PCR according to claim 2, wherein the oil phase channel and the sample channel have a T-shaped cross-flow structure and a Y-shaped cross-flow structure on the droplet chip structure, a cross-shaped flow focusing structure or a co-flow structure, the co-flow structure is that the oil phase channel is nested inside the sample channel.
A high-throughput integrated micro-droplet digital PCR implementation system according to claim 2, wherein the droplet chip is provided with several groups of micro-droplet generators, each group of micro-droplet generators Including the oil phase channel, the sample channel and the detection chamber, a single disturbance device is used to drive all the oil circuits or each oil circuit is provided with an independent disturbance device.
A system for implementing high-throughput integrated micro-droplet digital PCR according to claim 6, wherein a steering valve is provided at the outlet of each oil circuit, and the steering valve is connected to the oil circuit, the air pump and the oil circuit. phase channel.
A system for realizing high-throughput integrated micro-droplet digital PCR according to claim 2, wherein a blocking mechanism is provided at the entrance of the detection chamber, and a blocking mechanism is provided at the exit of the detection chamber. blocking mechanism.
A high-throughput integrated micro-droplet digital PCR implementation system according to claim 1, wherein the driving signal generated by the driver is a triangle wave, a rectangular wave or a sine wave; the generation frequency of the micro-droplet The frequency is consistent with the driving signal generated by the driver; the generation frequency of the micro-droplets is greater than 100 per second.
A high-throughput integrated micro-droplet digital PCR implementation system according to any one of claims 1-9, wherein the micro-droplet digital PCR implementation system includes GUI and data analysis modules, GUI and The data analysis module controls the operation of the realization system of micro-droplet digital PCR.