WO2023060544A1 - 一种用于dPCR集成微流控芯片的PCR扩增机构 - Google Patents

一种用于dPCR集成微流控芯片的PCR扩增机构 Download PDF

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WO2023060544A1
WO2023060544A1 PCT/CN2021/124043 CN2021124043W WO2023060544A1 WO 2023060544 A1 WO2023060544 A1 WO 2023060544A1 CN 2021124043 W CN2021124043 W CN 2021124043W WO 2023060544 A1 WO2023060544 A1 WO 2023060544A1
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temperature
flow channel
circulation
droplet
preheating
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PCT/CN2021/124043
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English (en)
French (fr)
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裴颢
郑文山
龚劲劲
黄兴
刘林波
吴翠
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墨卓生物科技(浙江)有限公司
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Priority to PCT/CN2021/124043 priority Critical patent/WO2023060544A1/zh
Publication of WO2023060544A1 publication Critical patent/WO2023060544A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/36—Apparatus for enzymology or microbiology including condition or time responsive control, e.g. automatically controlled fermentors
    • C12M1/38—Temperature-responsive control
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]

Definitions

  • the invention relates to the technical field of biological detection, and more specifically, relates to a PCR amplification mechanism for dPCR integrated microfluidic chips.
  • digital droplet PCR technology mainly includes three separate technologies, namely droplet generation technology, PCR thermal cycle amplification technology and droplet detection technology.
  • the PCR amplification is used to cycle the droplet containing the sample solution to heat up and down, so as to meet the temperature and duration requirements required by the PCR amplification.
  • the standard PCR process usually takes several hours to complete, and due to the limitation of PCR amplification, more time is required for detection.
  • the present invention aims to provide a PCR amplification mechanism for dPCR integrated microfluidic chips.
  • the present invention adopts the following technical solutions:
  • the embodiment of the present invention discloses a PCR amplification mechanism for a dPCR integrated microfluidic chip, including a circulation channel,
  • the circulation channel includes a plurality of high-temperature sections and a plurality of low-temperature sections arranged at intervals, so that the liquid droplets undergo a high-temperature-low temperature...high-temperature-low temperature circulation flow in the circulation channel;
  • the end of the circulation channel is connected to the liquid drop detection mechanism.
  • the preheating flow path also includes a preheating flow path, the beginning of the preheating flow path is connected to the end of the droplet output pipeline, the end of the preheating flow path is connected to the beginning of the circulation flow path, and the preheating flow path
  • the channel is used to heat the droplet to a first predetermined temperature.
  • the circulation channel includes a plurality of single circulation channels connected end to end in sequence, and each single circulation channel includes a low-temperature section and a high-temperature section.
  • circulation channel is integrally formed.
  • preheating runner and the circulation runner are integrally formed.
  • the multiple single-circulation channels have the same length and are arranged in parallel on the same plane, so that the multiple low-temperature sections form a low-temperature zone, and the multiple high-temperature sections form a high-temperature zone.
  • the first temperature control device controls the temperature of the preheating runner and the high temperature section
  • the second temperature control device controls the temperature of the low temperature section segment temperature
  • the first temperature control device includes a first heating sheet, and the first heating sheet is mounted on the high temperature section of the preheating flow path and the circulation flow path;
  • the second temperature control device includes a second heating chip, and the second heating chip is mounted on the low-temperature section of the circulation channel.
  • the preheating runner and the circulation flow channel are arranged on the backing plate, and,
  • a first heating sheet is arranged between the high temperature section and the preheating runner and the backing plate, and a second heating sheet is arranged between the low temperature section and the backing plate.
  • the present invention realizes rapid amplification of droplets by preheating tiny droplets to a first predetermined temperature, and then carrying out high temperature-low temperature...high temperature-low temperature circulation flow in the circulation flow channel.
  • the amplification time is shortened, and the detection efficiency is greatly improved under the premise of ensuring the accuracy.
  • Fig. 1 is the structural representation of an embodiment of the present invention
  • Fig. 2 is the structural representation of another embodiment of the present invention.
  • FIG. 3 is a schematic diagram of a two-phase confluence node in an embodiment of the present invention.
  • FIG. 4 is a schematic diagram of a droplet generating mechanism in an embodiment of the present invention.
  • Fig. 5 is a state diagram when droplets are generated according to an embodiment of the present invention.
  • FIG. 6 is a schematic flow channel diagram of a PCR amplification mechanism according to an embodiment of the present invention.
  • Fig. 7 and Fig. 8 are schematic structural diagrams of a dPCR integrated microfluidic chip according to an embodiment of the present invention.
  • FIG. 9 is a schematic structural diagram of a liquid drop detection mechanism according to an embodiment of the present invention.
  • some embodiments of the present invention disclose a dPCR integrated microfluidic chip, which includes a droplet generation mechanism 1 , a PCR amplification mechanism 2 and a droplet detection mechanism 3 connected in sequence.
  • the droplet generation mechanism 1, the PCR amplification mechanism 2 and the droplet detection mechanism 3 are sequentially connected, so that the entire device is integrated, and the detection error caused by the transfer of the detection sample between the instruments is eliminated. , also reduces manual operations, and greatly shortens the time required for detection.
  • the droplet generating mechanism 1 includes a water phase delivery pipeline 11, an oil phase delivery pipeline 12 and a droplet output pipeline 13, the end of the water phase delivery pipeline 11, the oil phase delivery pipeline The ends of 12 are all connected to the beginning of the droplet output pipeline 13, and a two-phase confluence node 14 is formed at the connection, as shown in Figure 3;
  • a water phase flow rate control mechanism is provided at the beginning of the water phase delivery pipeline 11
  • an oil phase flow rate control mechanism is provided at the beginning of the oil phase delivery pipeline 12, wherein the water phase flow rate control mechanism and the The oil phase flow rate control mechanism can be realized by pressure control or flow control, as shown in Figure 4.
  • a water-phase pressure control mechanism is provided at the beginning of the water-phase delivery pipeline 11, and an oil-phase pressure control mechanism is arranged at the beginning of the oil-phase delivery pipeline 12, so that the water-phase and oil-phase pressures are adjusted respectively.
  • Pressure control, the pressures of the water phase and the oil phase are individually adjusted, so that the size of the resulting initial droplets 4 can be adjusted.
  • the droplet size obtained in the embodiment of the present invention can be between 28-110 microns, and the droplet size can be adjusted as required. Under the same volume of samples, compared with the prior art, the present invention can generate about 1-16 times the number of droplets, achieve higher detection accuracy, and can detect samples with lower concentrations.
  • the droplet size obtained by the experiment is 100-110um, because the droplet generation scheme is to add a negative pressure at the droplet outlet, resulting in the separation of the water phase and the oil phase. The pressure value is the same.
  • the pressure of the water phase delivery pipeline 11 is the same as the pressure of the oil phase delivery pipeline 12
  • the pressure ratio is (0.9-1.1):1; and/or, the pressure range of the water phase delivery pipeline 11 is 200mbar-1500mbar.
  • the size, generation rate and flow velocity of the initial droplet 4 can be adjusted by adjusting the relationship between the flow rate of the oil phase and the water phase.
  • the control scheme is as follows: increasing the oil phase flow rate can reduce the droplet size and increase the droplet flow velocity; increasing the water phase flow rate can increase the droplet generation rate: increasing the water phase and oil phase flow rates simultaneously can keep the droplet size constant and increase the flow rate. speed.
  • a dPCR integrated microfluidic chip disclosed in some embodiments of the present invention on the basis of the above embodiments, in the droplet generating mechanism 1, the water phase delivery pipeline 11, the oil phase delivery pipeline 12 and the droplet output pipeline 13 integrally formed.
  • the oil phase pressure control mechanism is a gas pressurization control mechanism.
  • the oil phase pressure control mechanism includes an oil phase gas pump 121, and the oil phase gas pump 121 is arranged at the beginning of the oil phase delivery pipeline 12:
  • the water phase pressure control mechanism includes a water phase gas pump 111 , the water phase gas pump 111 is arranged at the beginning of the water phase delivery pipeline.
  • the oil phase pressure control mechanism also includes an oil phase gas pipe 122, the two ends of the oil phase gas pipe 122 are respectively connected to the beginning of the oil phase gas pump 121 and the oil phase delivery pipeline 12; the water phase pressure control mechanism also A water phase gas pipe 112 is included, and the two ends of the water phase gas pipe 112 are respectively connected to the water phase gas pump 111 and the beginning of the water phase delivery pipeline 11 .
  • the pressures of the water phase and the oil phase are controlled by the water phase gas pump 111 and the oil phase gas pump 121, so as to realize the independent control of the flow rate and flow rate of the water phase and the oil phase, so that the liquid droplets can better meet the detection requirements.
  • air pressure controllers 15 are respectively provided on the water phase gas pipe and the oil phase gas pipe to precisely control the air pressure.
  • a dPCR integrated microfluidic chip disclosed in some embodiments of the present invention on the basis of the above embodiments, in the droplet generating mechanism 1, at the two-phase confluence node 14, the water-phase delivery pipeline 11 is connected to the The centerlines of the droplet delivery pipelines are on the same straight line.
  • the oil phase delivery pipeline 12 forms a first included angle with the centerline of the droplet delivery pipeline.
  • the first included angle is preferably a right angle.
  • the droplet generation mechanism is the interaction of two incompatible liquid phases, one of which enters the other phase and is sheared or split into droplets due to instability. When carrying out liquid transportation, the oil phase can be cut off from the vertical direction, which can better realize the segmentation of the water phase, so as to obtain smaller initial droplets 4 .
  • the oil phase delivery pipeline 12 includes an oil phase delivery main pipe and at least two oil phase As for the delivery branch pipe, the beginning ends of at least two oil phase delivery branch pipes are connected in parallel to the end of the oil phase delivery main pipe, and the ends of at least two oil phase delivery branch pipes are connected to the beginning end of the droplet output pipeline 13 . That is, there may be multiple oil phase delivery branch pipes. In some preferred embodiments, the ends of the multiple oil phase delivery branch pipes may be evenly arranged at the two-phase confluence node 14 in the circumferential direction.
  • the oil phase delivery branch pipe may include a first oil phase delivery branch pipe 124 and a second oil phase delivery branch pipe 125 .
  • the water phase delivery pipeline 11, the first oil phase delivery branch pipe 124, the second oil phase delivery branch pipe 125 and the droplet delivery pipeline are connected in a cross, and, The centerlines of the water phase delivery pipeline 11 and the droplet delivery pipeline are on the same straight line.
  • the oil phase pressure control mechanism can be arranged in the oil phase delivery main pipe, so that the pressure obtained by any oil phase delivery branch pipe is the same.
  • a dPCR integrated microfluidic chip disclosed in some embodiments of the present invention on the basis of the above embodiments, in the droplet generating mechanism 1, in order to stabilize the oil phase transport entering the two-phase confluence node 14, the oil
  • the phase conveying branch pipe is provided with a steady flow channel 123 near the end.
  • the PCR amplification mechanism 2 includes a sequentially connected preheating flow channel and a circulation flow channel 22,
  • the preheating channel is used to heat the droplet to a first predetermined temperature
  • the circulation channel 22 includes a plurality of high-temperature sections 223 and a plurality of low-temperature sections 224 arranged at intervals, so that the liquid droplets can circulate in the circulation channel 22 at high temperature-low temperature...high temperature-low temperature;
  • the end of the circulation channel 22 is connected to the drop detection mechanism 3 .
  • the PCR amplification mechanism 2 is used to cycle the droplet containing the sample solution, that is, the initial droplet 4, to heat up and down in a cycle, so as to meet the temperature requirement and duration requirement required by the PCR amplification.
  • the rapid expansion of droplets is realized by preheating the tiny droplets to the first predetermined temperature, and then performing a high temperature-low temperature...high temperature-low temperature circulation flow in the circulation flow channel 22. Amplification is complete within 20-30 minutes. In the prior art, the standard PCR process usually takes several hours to complete.
  • the circulation flow channel 22 includes a plurality of single circulation flow channels 221 connected end to end in sequence, each of the single circulation flow channels 221
  • the circulation channel 221 includes a low temperature section 224 and a high temperature section 223 .
  • the ends of two adjacent single-circulation flow channels 221 are connected by bending flow channels 222 .
  • the circulation channel 22 is integrally formed.
  • the preheating runner and the circulation runner 22 are integrally formed.
  • the water phase delivery pipeline 11 , the oil phase delivery pipeline 12 , the droplet output pipeline 13 , the preheating flow channel and the circulation flow channel 22 are integrally formed.
  • the PCR amplification mechanism 2 also includes a first temperature control device and a second temperature control device, the first The temperature control device controls the temperature of the preheating runner and the high temperature section 223 , and the second temperature control device controls the temperature of the low temperature section 224 . Accurate temperature control is realized by the first temperature control device and the second temperature control device, thereby further ensuring smooth and fast amplification.
  • the dPCR integrated microfluidic chip of the present invention can also be set with more than two temperature control areas, such as low temperature...medium temperature...high temperature circulation can be set to flow to complete the expansion. increase.
  • the PCR amplification mechanism 2 also includes a backing plate 25, the preheating flow channel and the circulation flow channel 22 is placed on the backing plate 25, and the first heating sheet 23 is set between the high temperature section 223 and the preheating runner and the backing plate 25, and the first heating sheet 23 is set between the low temperature section 224 and the backing plate 25.
  • the preheating flow channel like the circulation flow channel 22, is set as a serpentine flow channel, which can make the single flow channel in the serpentine flow channel
  • the length is not greater than the length of the high temperature section 223, so that the first heating sheet 23 and the second heating sheet 24 can be arranged as a rectangle that is easy to process and control.
  • the heating areas of the heating chips 24 interfere with each other.
  • the multiple single-circulation channels 221 have the same length and are arranged in parallel on the same plane, so that the multiple low-temperature sections 224 form a low-temperature zone 226 , and the multiple high-temperature sections 223 form a high-temperature zone 225 .
  • the first temperature control device includes a first heating chip 23, and the first heating chip 23 is mounted on the preheating runner and the circulation on the high temperature section 223 of the flow channel 22 ;
  • the second temperature control device includes a second heating chip 24 , and the second heating chip 24 is mounted on the low temperature section 224 of the circulation flow channel 22 .
  • the preheating flow channel and the circulation flow channel 22 can be set to a structure capable of lateral displacement on the backing plate 25, so that the high temperature section 223 and the low temperature section 224
  • the length can be adjusted according to needs.
  • the width of the preheating runner should be smaller than the length of the high temperature section 223, so as to ensure that the preheating runner will not be affected during the moving process and meet the requirements of more accurate amplification temperature, thereby achieving more Excellent amplification speed.
  • the control scheme is as follows: the temperature of the high and low temperature zone 226 can be adjusted separately through the external hot plate; the retention time of the droplet at each temperature can be controlled by the flow rate of the droplet and the length of the flow channel; the number of cycles can be controlled by changing the flow rate.
  • the cycle number of the channel can be adjusted.
  • the temperature of the circulation of the droplets, the holding time at each temperature and the number of cycles are all controllable. It can be adjusted by adjusting the flow rate and the size of the channel structure according to the requirements of PCR amplification.
  • the flow rate can be decreased and the channel size can be increased, and vice versa.
  • the height of the detection channel of the droplet detection mechanism 3 at the detection positioning point is smaller than the diameter of the droplet to be detected, so as to ensure the detection of each droplet. Same thickness.
  • An optical inspection system is installed at the position corresponding to the detection positioning point.
  • Some embodiments of the present invention also disclose a droplet detection method using a dPCR integrated chip
  • the speed of the droplet to be detected passing through the droplet detection mechanism is controlled by respectively controlling the flow rates of the oil phase and the water phase entering the droplet generating mechanism.
  • a water phase pressure control mechanism is arranged at the beginning of the water phase conveying pipeline, and an oil phase pressure control mechanism is arranged at the beginning of the oil phase conveying pipeline, so that positive pressure control of the water phase and the oil phase pressure is realized respectively , the pressures of the water and oil phases are individually adjusted, making the size of the resulting initial droplets adjustable.
  • Some embodiments of the present invention disclose a droplet detection method using a dPCR integrated chip. On the basis of the above-mentioned embodiments, the initial droplet size and The distance between two adjacent droplets to be detected.
  • Some embodiments of the present invention disclose a liquid drop detection method using a dPCR integrated chip.
  • the height of the detection channel at the detection positioning point is smaller than the diameter of the liquid drop to be detected, so as to ensure that each Droplets are detected at the same thickness.
  • Some embodiments of the present invention disclose a droplet detection method using a dPCR integrated chip.
  • amplifying the initial droplet through a PCR amplification mechanism includes:
  • the initial droplets After the initial droplets are preheated to the first predetermined temperature through the preheating flow channel, they enter the circulation flow channel, and carry out high temperature-low temperature...high temperature-low temperature circulation flow in the circulation flow channel to obtain amplified droplets.
  • Generation of initial droplets by the droplet generation mechanism includes:
  • the residence time of the initial droplets in the PCR amplification mechanism is 20-30 minutes.
  • the size of the preheating flow channel and the circulation flow channel is determined by the required retention time and flow velocity of the liquid droplets in the flow channel.
  • the flow rate of the liquid droplets in the flow channel is further adjusted by controlling the flow keep time.
  • the high temperature is 80-99°C or 88-98°C; and/or,
  • the temperature of the low temperature is 45-75°C or 60-65°C.
  • the area where the droplet passes is divided into three areas.
  • the first area is the droplet preheating area.
  • the droplet will be heated from room temperature to 95°C, and then enter the temperature cycle area, including the high temperature area of 95°C and the low temperature area of 60°C.
  • the droplets circulate back and forth in the flow channel, thereby realizing the amplification of DNA.
  • Droplets can quickly change between high and low temperatures when they flow in the flow channel, breaking through the long time-consuming problem of heating and cooling of the hot plate in the traditional heating method.
  • the residence time of droplets in each temperature zone can be controlled by adjusting the air pressure of the pneumatic pump according to demand.
  • the left and right length of a flow channel is 53 mm, and the time for droplets to pass can be controlled between 5 and 60 seconds.
  • Some embodiments of the present invention disclose a droplet detection method using a dPCR integrated chip.
  • the ratio of the pressure at the inlet of the oil phase to the pressure at the inlet of the water phase is (0.9-1.1):1.
  • the pressure range at the inlet of the water phase is 200mbar-1500mbar.
  • the circulation channel includes a plurality of single circulation channels connected end to end in sequence, each of the single circulation channels includes a low temperature section and a high temperature section, and multiple The low-temperature section forms a low-temperature zone, and a plurality of the high-temperature sections form a high-temperature zone; the temperature of the high-temperature zone and the preheating runner is controlled by the first temperature control device, and the temperature of the low-temperature zone is controlled by the second temperature control device.
  • the detection flow channel is used to arrange the liquid droplets in a single row and pass through the flow channel after a certain distance from each other, so that An optical detection system is used to count the number of positive and negative droplets flowing through the channel.
  • the positive droplet refers to that the droplet contains target DNA, and after cyclic amplification, the droplet contains a large amount of target DNA, and the double-stranded DNA will combine with the fluorophore in the droplet to generate fluorescence.
  • Negative droplets are non-fluorescent.
  • This flow channel can control the passing speed of the droplets and the distance between the droplets, and can ensure that the signal intensity of the fluorescent droplets is consistent. .
  • the control scheme is as follows: the speed of the liquid droplets passing through the detection area can be regulated by controlling the flow rate of the oil phase. The greater the speed of the oil phase, the faster the speed of the liquid droplets passing through the detection area; the distance between the liquid droplets can be controlled by The flow ratio of the oil phase inlet and the droplet inlet is controlled by the flow ratio of the two phases.
  • the larger the flow rate at the oil phase inlet, the larger the distance between the droplets; in order to keep the signals of the fluorescent droplets consistent, we set the The height is designed to be smaller than the diameter of the droplet, so each droplet is compressed to the same height as the flow channel when passing through the channel, and the thickness of the droplet in the Z-axis direction is the same, so when detecting in the Z-axis direction , the fluorescence intensity of the droplet is not affected by the droplet thickness.
  • the volume of each sample solution is 20uL, the DNA concentration is 2-fold gradient dilution respectively, the DNA concentration range is 1000-10copies/uL, and a blank control group is added.
  • Detection results Compared with Bio-Rad's chip, the detection accuracy is the same, and the detection speed is 12 times faster than Bio-Rad's chip.
  • the detection speed can be increased by 12 times.
  • it takes 4 hours for three instruments of the model Bio-Rad brand QX-200 series to perform detection, but with the chip and detection technology of the present invention, the same detection only takes 20 minutes.
  • the detection accuracy is 0.25copies/uL.

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Abstract

本发明提供一种用于dPCR集成微流控芯片的PCR扩增机构，其属于生物检测技术领域。该PCR扩增机构包括顺序连通的预热流道和循环流道，其中，该预热流道用于使液滴加热至第一预定温度；该循环流道包括间隔设置的多个高温段和多个低温段，使液滴在该循环流道内进行高温-低温……高温-低温的循环流动：该预热流道的始端连通该液滴输出管道的末端；该循环流道的末端连通至液滴检测机构。本发明通过使微小液滴先预热道第一预定温度，然后在该循环流道内进行高温-低温……高温-低温的循环流动，实现了液滴的快速扩增。缩短了扩增时间，在保证准确性的前提下，极大地提高了检测效率。

Description

一种用于dPCR集成微流控芯片的PCR扩增机构 技术领域
本发明涉及生物检测技术领域，并且更具体地，涉及到一种用于dPCR集成微流控芯片的PCR扩增机构。
背景技术
对于数字液滴PCR技术，主要包含三个单独的技术，即液滴生成技术、PCR热循环扩增技术与液滴检测技术。
PCR扩增用于将包含样本溶液的液滴进行循环升降温，从而达到PCR扩增所要求的温度要求与持续时间要求。现有技术中，标准的PCR流程通常需要几个小时的时间才能完成，由于PCR扩增的限制，导致检测需要较多的时间。
基于此，现有技术仍然有待改进。
发明内容
本发明针对上述问题，目的在于提供一种用于dPCR集成微流控芯片的PCR扩增机构。
为达到上述目的，本发明采用如下技术方案：
本发明的实施例公开了一种用于dPCR集成微流控芯片的PCR扩增机构，包括循环流道，
所述循环流道包括间隔设置的多个高温段和多个低温段，使液滴在所述循环流道内进行高温-低温……高温-低温的循环流动；
所述循环流道的末端连通至液滴检测机构。
进一步地，还包括预热流道，所述预热流道的始端连通所述液滴输出管道的末端，所述预热流道的末端连接所述循环流道的始端，所述预热流道用于使液滴加热至第一预定温度。
进一步地，所述循环流道包括依次首尾相接的多个单循环流道，每个所述单循环流道包括低温段和高温段。
进一步地，相邻的两个所述单循环流道的首尾之间通过弯折流道连接。
进一步地，所述循环流道一体成型。
进一步地，所述预热流道和所述循环流道一体成型。
进一步地，多个所述单循环流道长度相同且平行布置在同一平面上，使多个所述低温段形成低温区，多个所述高温段形成高温区。
进一步地，还包括第一温度控制装置和第二温度控制装置，所述第一温度控制装置控制所述预热流道和所述高温段的温度，所述第二温度控制装置控制所述低温段的温度。
进一步地，所述第一温度控制装置包括第一加热片，所述第一加热片贴装在所述预热流道和所述循环流道的高温段上；
所述第二温度控制装置包括第二加热片，所述第二加热片贴装在所述循环流道的低温段上。
进一步地，还包括垫板，所述预热流道和所述循环流道安置在所述垫板上，并且，
所述高温段和所述预热流道与垫板之间设置有第一加热片，所述低温段与垫板之间设置有第二加热片。
本发明的有益效果是：
本发明通过使微小液滴先预热道第一预定温度，然后在所述循环流道内进行高温-低温……高温-低温的循环流动，实现了液滴的快速扩增。缩短了扩增时间，在保证准确性的前提下，极大地提高了检测效率。
附图说明
图1为本发明一实施例的结构示意图；
图2为本发明又一实施例的结构示意图；
图3为本发明一实施例中的两相汇流节点处的示意图；
图4为本发明一实施例中的液滴生成机构的示意图；
图5为本发明一实施例的液滴生成时的状态图；
图6为本发明一实施例的PCR扩增机构的流道示意图；
图7，图8为本发明一实施例的dPCR集成微流控芯片结构示意图；
图9为本发明一实施例的液滴检测机构处的结构示意图。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，下面结合附图及实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅用以解释本发明，并不用于限定本发明。
如图1-图9所示，本发明一些实施例公开了一种dPCR集成微流控芯片，其包括依次顺序连接的液滴生成机构1、PCR扩增机构2和液滴检测机构3。本实施例通过将液滴生成机构1、PCR扩增机构2和液滴检测机构3依次顺序连接，使得整个设备集成为一体，消除了因为检测样本在仪器之间的转移，而导致的检测误差，也减少了人工操作，并且大幅地缩短了检测所需要的时间。
在一些优选的实施例中，所述液滴生成机构1包括水相输送管道11、油相输送管道12和液滴输出管道13，所述水相输送管道11的末端、所述油相输送管道12的末端均连通至所述液滴输出管道13的始端，并在所述连通处形成两相汇流节点14，如图3所示；
并且，在所述水相输送管道11的始端设置有水相流速控制机构，在所述油相输送管道12的始端设置有油相流速控制机构，其中，所述水相流速控制机构和所述油相流速控制机构可以通过压力控制或流量控制来实现，如图4所示。
本发明通过在所述水相输送管道11的始端设置有水相压力控制机构，在所述油相输送管道12的始端设置有油相压力控制机构，实现了水相和油相压 力的分别正压控制，水相和油相的压力各自调节，使得得到的初始液滴4的尺寸可调节。
本发明实施例得到的液滴尺寸可以在28-110微米之间，且可以根据需要对液滴尺寸进行调节。在同体积样本下，与现有技术相比，本发明可以产生约1-16倍数量的液滴，达到更高的检测精度，可以实现检测更低浓度的样本。以Bio-Rad仪器(QX-200)为例，其实验得出的液滴尺寸为100-110um，由于其液滴生成方案是在液滴出口处加一个负压，导致水相和油相的压力值相同，当液滴生成的结构和尺寸确定之后，液滴尺寸就确定了，且调节负压大小对液滴大小影响不大，无法做到液滴尺寸的调整。
本发明一些实施例所公开的一种dPCR集成微流控芯片，在上述实施例的基础上，液滴生成机构1中，所述水相输送管道11的压力与所述油相输送管道12的压力比值为(0.9-1.1)：1；和/或，所述水相输送管道11的压力范围为200mbar-1500mbar。初始液滴4的大小、生成速率和流动速度可以通过调控油相和水相流量的关系来调整。调控方案为：增加油相流量可以降低液滴大小和提高液滴流动速度；增加水相流量可以提高液滴生成速率：同步增加水相和油相流量可以实现保持液滴大小不变，提高流动速度。
本发明一些实施例所公开的一种dPCR集成微流控芯片，在上述实施例的基础上，液滴生成机构1中，所述水相输送管道11、油相输送管道12和液滴输出管道13一体成型。
本发明一些实施例所公开的一种dPCR集成微流控芯片，在上述实施例的基础上，液滴生成机构1中，为了实现精确的压力控制，所述油相压力控制结构和所述水相压力控制机构为气体加压控制机构。一些优选的实施方式中，所述油相压力控制机构包括油相气泵121，所述油相气泵121设置在所述油相输送管道12的始端：所述水相压力控制机构包括水相气泵111，所述水相气泵111设置在所述水相输送管路的始端。所述油相压力控制机构还包括油相气管122，所述油相气管122的两端分别连接所述油相气泵121和所述油相输 送管道12的始端；所述水相压力控制机构还包括水相气管112，所述水相气管112的两端分别连接所述水相气泵111和所述水相输送管道11的始端。通过水相气泵111和油相气泵121分别对水相和油相的压力进行控制，从而实现水相和油相的流量和流速的分别单独控制，进而使液滴更能满足检测需求。进一步地，在水相气管和油相气管上分别设置气压控制器15，以精确控制气压。
本发明一些实施例所公开的一种dPCR集成微流控芯片，在上述实施例的基础上，液滴生成机构1中，所述两相汇流节点14处，所述水相输送管道11与所述液滴输送管道的中心线在同一直线上。所述两相汇流节点14处，所述油相输送管道12与所述液滴输送管道的中心线呈第一夹角。所述第一夹角优选为直角。液滴的产生机理为两个不相容的液相相互作用，其中一相进入另外一相被剪切或者由于不稳定而分裂成液滴。进行液体输送时，油相可由垂直方向切下，可更好地实现对水相的分割，从而获得尺寸更小的初始液滴4。
本发明一些实施例所公开的一种dPCR集成微流控芯片，在上述实施例的基础上，液滴生成机构1中，所述油相输送管道12包括油相输送总管和至少两个油相输送支管，至少两个所述油相输送支管的始端并联连接至所述油相输送总管的末端，至少两个所述油相输送支管的末端连通至所述液滴输出管道13的始端。即油相输送支管可以为多个，在一些优选的实施方式中，多个油相输送支管的末端可周向均匀地布置在两相汇流节点14处。
具体地，如图4所示，所述油相输送支管可包括第一油相输送支管124和第二油相输送支管125。所述两相汇流节点14处，所述水相输送管道11、所述第一油相输送支管124、所述第二油相输送支管125和所述液滴输送管道呈十字交叉连接，并且，所述水相输送管道11和所述液滴输送管道的中心线在同一直线上。所述油相压力控制机构可设置在油相输送总管，使得任一油相输送支管得到的压力相同。
本发明一些实施例所公开的一种dPCR集成微流控芯片，在上述实施例的基础上，液滴生成机构1中，为了使进入两相汇流节点14处的油相输送稳定， 所述油相输送支管在靠近末端处设置有稳流流道123。
本发明一些实施例所公开的一种dPCR集成微流控芯片，如图1和图6至图8所示，所述PCR扩增机构2包括顺序连通的预热流道和循环流道22，
其中，所述预热流道用于使液滴加热至第一预定温度；
所述循环流道22包括间隔设置的多个高温段223和多个低温段224，使液滴在所述循环流道22内进行高温-低温……高温-低温的循环流动；
所述预热流道的始端连通所述液滴输出管道13的末端：
所述循环流道22的末端连通至液滴检测机构3。
PCR扩增机构2用于将包含样本溶液的液滴即初始液滴4进行循环升降温，从而达到PCR扩增所要求的温度要求与持续时间要求。本实施例通过使微小液滴先预热道第一预定温度，然后在所述循环流道22内进行高温-低温……高温-低温的循环流动，实现了液滴的快速扩增，可在20-30分钟内完成扩增。而现有技术中，标准的PCR流程通常需要几个小时的时间才能完成。
本发明一些实施例所公开的一种dPCR集成微流控芯片，在上述实施例的基础上，所述循环流道22包括依次首尾相接的多个单循环流道221，每个所述单循环流道221包括低温段224和高温段223。相邻的两个所述单循环流道221的首尾之间通过弯折流道222连接。
本发明一些实施例所公开的一种dPCR集成微流控芯片，在上述实施例的基础上，所述循环流道22一体成型。一些实施例中，所述预热流道和所述循环流道22一体成型。还有一些实施例中，水相输送管道11、油相输送管道12、液滴输出管道13、所述预热流道和所述循环流道22一体成型。
本发明一些实施例所公开的一种dPCR集成微流控芯片，在上述实施例的基础上，所述PCR扩增机构2还包括第一温度控制装置和第二温度控制装置，所述第一温度控制装置控制所述预热流道和所述高温段223的温度，所述第二温度控制装置控制所述低温段224的温度。通过第一温度控制装置和第二温度控制装置实现精确的温度控制，从而进一步保证扩增的顺利快速进行。
需要指出的是，本发明的dPCR集成微流控芯片，基于PCR扩增的需要，也可以设置多于两个温度控制区域，如可以设置为低温…中温…高温循环的方式进行流动来完成扩增。
本发明一些实施例所公开的一种dPCR集成微流控芯片，在上述实施例的基础上，所述PCR扩增机构2还包括垫板25，所述预热流道和所述循环流道22安置在所述垫板25上，并且，所述高温段223和所述预热流道与垫板25之间设置有第一加热片23，所述低温段224与垫板25之间设置有第二加热片24。即加热片位于垫板25和流道之间。在一些实施方式中，为了方便第一加热片23和第二加热片24的布置，预热流道与循环流道22一样，设置成蛇形流道，可以使蛇形流道中单个流道的长度不大于高温段223的长度，使得第一加热片23和第二加热片24可以设置为容易加工和控制的矩形，同时，还能尽可能地避免第一加热片23的加热区域与第二加热片24的加热区域互相干扰。
多个所述单循环流道221长度相同且平行布置在同一平面上，使多个所述低温段224形成低温区226，多个所述高温段223形成高温区225。
本发明一些优选的实施方式，在上述实施例的基础上，所述第一温度控制装置包括第一加热片23，所述第一加热片23贴装在所述预热流道和所述循环流道22的高温段223上；所述第二温度控制装置包括第二加热片24，所述第二加热片24贴装在所述循环流道22的低温段224上。
本发明一些优选的实施方式，在上述实施例的基础上，可将预热流道和循环流道22设置成可以在垫板25上进行横向位移的结构，使得高温段223和低温段224的长度可以根据需要进行调整，此时，预热流道的宽度应小于高温段223的长度，保证在移动过程中预热流道不会受到影响，满足更精确的扩增温度要求，从而实现更佳的扩增速度。
控制方案为：高低温区226的温度可以通过外部热板进行分别调控；液滴在每个温度下的保持时间可以通过液滴的流速与流道的长短来控制；循环数则可以通过改变流道的循环数即可调整。上述实施例提供的PCR扩增机构2， 液滴的循环温度的高低、每个温度下的保持时间和循环数都可控。可以根据PCR扩增的要求通过调整流速和流道结构尺寸来调整。
具体而言即需要增加保持时间时，可降低流速与增长流道尺寸，反之亦然。
本发明一些优选的实施方式，在上述实施例的基础上，所述液滴检测机构3在检测定位点处的检测流道的高度小于待检测液滴的直径，以保证每个液滴的检测厚度相同。所述检测定位点相对应的位置设置有光学检查系统。
本发明一些实施例还公开了一种采用dPCR集成芯片的液滴检测方法，
通过液滴生成机构生成初始液滴；
将所述初始液滴通过PCR扩增机构进行扩增，得到扩增液滴；
将所述扩增液滴通过液滴检测机构排列处理后得到待检测液滴，并对所述待检测液滴进行光学检测；
其中，通过分别控制进入液滴生成机构的油相和水相的流速控制待检测液滴通过液滴检测机构的速度。
本发明通过在所述水相输送管道的始端设置有水相压力控制机构，在所述油相输送管道的始端设置有油相压力控制机构，实现了水相和油相压力的分别正压控制，水相和油相的压力各自调节，使得得到的初始液滴的尺寸可调节。
本发明一些实施例公开的一种采用dPCR集成芯片的液滴检测方法，在上述实施例的基础上，通过控制进入液滴生成机构的油相和水相的流量比控制初始液滴的大小和相邻两个待检测液滴之间的间距。
本发明一些实施例公开的一种采用dPCR集成芯片的液滴检测方法，在上述实施例的基础上，在检测定位点处的检测流道的高度小于待检测液滴的直径，以保证每个液滴的检测厚度相同。
本发明一些实施例公开的一种采用dPCR集成芯片的液滴检测方法，在上述实施例的基础上，将所述初始液滴通过PCR扩增机构进行扩增包括：
将初始液滴经过预热流道预热至第一预定温度后，进入循环流道，在循环流道内进行高温-低温……高温-低温的循环流动，得到扩增液滴。
通过液滴生成机构生成初始液滴包括：
分别在油相入口和水相入口处加正压，使油相和水相在预定压力下在两相汇流节点处汇流，得到初始液滴。
所述初始液滴在所述PCR扩增机构中的停留时间为20-30分钟。所述预热流道和所述循环流道的尺寸由液滴在流道内需要的保持时间和流速决定，在使用中通过控制水相和油相的流量来进一步调节液滴在流道内需要的保留时间。
所述循环流道内，高温的温度为80-99℃或88-98℃；和/或，
所述循环流道内，低温的温度为45-75℃或60-65℃。
即液滴经过的区域分为三个区域，第一个区域是液滴预热区，液滴会从常温加热到95℃，然后进入温度循环区，包括高温区95℃和低温区60℃，液滴在流道中来回循环流动，进而实现了DNA的扩增。液滴在流道中流动即可快速在高低温下变化，突破了传统的加热方式中热板的加热与冷却的长耗时问题。每个温区液滴停留的时间可以根据需求调节气压泵的气压来控制，一些实施方式中，一个流道左右长度为53毫米，液滴通过的时间可以在5～60秒之间控制，一共30条流道，即1个循环可以控制在5～60秒之间(也可以更慢到准静态流动)，30个循环。其扩增速度远远高于采用单个温控板升降温(一个升降温循环最快需要180秒)的扩增速度。
本发明一些实施例公开的一种采用dPCR集成芯片的液滴检测方法，在上述实施例的基础上，初始液滴生成时，油相入口处的压力与所述水相入口处的压力比为(0.9-1.1)：1。所述水相入口处的压力范围为200mbar-1500mbar。
本发明一些实施例所公开的dPCR集成芯片及检测方法，所述循环流道包括依次首尾相接的多个单循环流道，每个所述单循环流道包括低温段和高温段，多个所述低温段形成低温区，多个所述高温段形成高温区；通过第一温 度控制装置控制高温区和预热流道的温度，通过第二温度控制装置控制低温区的温度。
本发明一些实施例所公开的dPCR集成芯片及检测方法，如图9所示，在液滴检测阶段，检测流道用于将液滴排成单列且相互间隔一定距离后通过该流道，以便于光学检测系统来统计流过该流道的阳性液滴和阴性液滴的数量。
阳性液滴指的是液滴中包含目标DNA，然后经过循环扩增后液滴中包含了大量的目标DNA，双链DNA会与液滴中的荧光基团结合而产生荧光。阴性液滴则是没有荧光。
其结构与液滴生成机构相似，也是从侧部通入油相，该流道可以控制液滴的通过速度与液滴之间的间距，并且可以保证有荧光的液滴的信号强度都保持一致。目前市场上没有产品中使用过类似流道。
调控方案为：液滴通过检测区的速度可以通过控制油相的流速来调控，油相加入的速度越大，则液滴通过检测区的速度就越快；液滴之间的间距可以通过控制油相入口与液滴入口两相的流量比值来控制，油相入口处的流量越大，液滴的间距就越大；为了保持有荧光的液滴的信号都保持一致，我们把流道的高度设计为比液滴直径小，所以每一个液滴在经过该流道时都被压缩成和流道高度一样高，则液滴Z轴方向上的厚度一样，所以在Z轴方向上检测时，液滴的荧光强度不会受液滴厚度影响。
实施例
配置检测标准溶液：
每个样本液体积20uL，DNA浓度分别为以2倍梯度稀释，DNA浓度范围在1000-10copies/uL，另加一组空白对照组。
检测结果：与Bio-Rad的芯片相比，检测准确性相同，检测速度比Bio-Rad的芯片快12倍。
综上所述，采用本发明设计的芯片，检测速度可以提高12倍。现有技术中，例如型号Bio-Rad品牌QX-200系列三台仪器做检测需要时间4小时，采用 本发明的芯片和检测技术，同样的检测只需要20分钟。检测精度0.25copies/uL。
以上所述仅为本发明的较佳实施例，并非用来限定本发明的实施范围；如果不脱离本发明的精神和范围，对本发明进行修改或者等同替换，均应涵盖在本发明权利要求的保护范围当中。

Claims (9)

  1. 一种用于dPCR集成微流控芯片的PCR扩增机构，其特征在于，
    包括循环流道，
    所述循环流道包括间隔设置的多个高温段和多个低温段，使液滴在所述循环流道内进行高温-低温……高温-低温的循环流动；
    所述循环流道的末端连通至液滴检测机构。
  2. 根据权利要求1所述的PCR扩增机构，其特征在于，还包括预热流道，所述预热流道的始端连通所述液滴输出管道的末端，所述预热流道的末端连接所述循环流道的始端，所述预热流道用于使液滴加热至第一预定温度。
  3. 根据权利要求1或2所述的PCR扩增机构，其特征在于，所述循环流道包括依次首尾相接的多个单循环流道，每个所述单循环流道包括低温段和高温段。
  4. 根据权利要求3所述的PCR扩增机构，其特征在于，相邻的两个所述单循环流道的首尾之间通过弯折流道连接；
    和/或，所述循环流道一体成型。
  5. 根据权利要求2所述的PCR扩增机构，其特征在于，所述预热流道和所述循环流道一体成型。
  6. 根据权利要求3或4所述的PCR扩增机构，其特征在于，多个所述单循环流道长度相同且平行布置在同一平面上，使多个所述低温段形成低温区，多个所述高温段形成高温区。
  7. 根据权利要求1-6至少一项所述的PCR扩增机构，其特征在于，还包括预热流道，所述预热流道的始端连通所述液滴输出管道的末端，所述预热流道的末端连接所述循环流道的始端，所述预热流道用于使液滴加热至第一预定温度，还包括第一温度控制装置和第二温度控制装置，所述第一温度控制装置控制所述预热流道和所述高温段的温度，所述第二温度控制装置控制所述低温段的温度。
  8. 根据权利要求7所述的PCR扩增机构，其特征在于，所述第一温度控制装置包括第一加热片，所述第一加热片贴装在所述预热流道和所述循环流道的高温段上；
    所述第二温度控制装置包括第二加热片，所述第二加热片贴装在所述循环流道的低温段上。
  9. 根据权利要求2或5所述的PCR扩增机构，其特征在于，还包括垫板，所述预热流道和所述循环流道安置在所述垫板上，并且，
    所述高温段和所述预热流道与垫板之间设置有第一加热片，所述低温段与垫板之间设置有第二加热片。
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