WO2021056651A1 - 超高通量单细胞核酸分子实时荧光定量分析方法、芯片和系统 - Google Patents

超高通量单细胞核酸分子实时荧光定量分析方法、芯片和系统 Download PDF

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WO2021056651A1
WO2021056651A1 PCT/CN2019/112903 CN2019112903W WO2021056651A1 WO 2021056651 A1 WO2021056651 A1 WO 2021056651A1 CN 2019112903 W CN2019112903 W CN 2019112903W WO 2021056651 A1 WO2021056651 A1 WO 2021056651A1
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fluorescence
axis
chip
sample
nucleic acid
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PCT/CN2019/112903
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English (en)
French (fr)
Inventor
周连群
张芷齐
张威
李传宇
李金泽
郭振
姚佳
李超
Original Assignee
中国科学院苏州生物医学工程技术研究所
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Priority claimed from CN201910911821.3A external-priority patent/CN110734854B/zh
Priority claimed from CN201910912751.3A external-priority patent/CN110643688A/zh
Priority claimed from CN201910912693.4A external-priority patent/CN110628567B/zh
Application filed by 中国科学院苏州生物医学工程技术研究所 filed Critical 中国科学院苏州生物医学工程技术研究所
Publication of WO2021056651A1 publication Critical patent/WO2021056651A1/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
    • C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
    • 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
    • C12Q1/6851—Quantitative amplification

Definitions

  • This application relates to the technical field of gene detection, in particular to an ultra-high-throughput single-cell nucleic acid molecule real-time fluorescence quantitative analysis method, chip and system.
  • the growth, development, differentiation, aging and pathological changes of the body are all related to the differential expression of genes.
  • the occurrence, development and metastasis of tumors are also related to mutations and differential expression of genes.
  • Cells in the center of tumor tissues, surrounding cells, and metastatic cells, etc. are also due to differences in genome and transcriptional expression profiles, resulting in different functional characteristics, affecting and Determine the results of tumor treatment.
  • RT-PCR real-time PCR
  • RT-qPCR fluorescent quantitative RT-PCR
  • a single-cell multi-gene detection system came into being. It usually uses microfluidic channels to make independent units, isolates single cells in independent units, and performs cDNA amplification. At most hundreds to thousands of single cells are captured each time, and then the instrument is used for nucleic acid amplification.
  • the number of micropore channels of the existing microfluidic chip is generally only a few hundred to tens of thousands. The number of single cells captured each time is small, and the detection throughput is low, which cannot meet the needs of ultra-high-throughput cell nucleic acid molecule detection. .
  • the current single-cell analysis methods have defects such as low throughput, and they need to be combined with nucleic acid fluorescence real-time quantitative analysis instruments.
  • the existing nucleic acid real-time quantitative analysis instrument is based on the analysis of the nucleic acid molecule of the population cell, and it loses the information of cell heterogeneity and the key information of the functional diversity of single cells. So now it is necessary to provide a more reliable solution.
  • the technical problem to be solved by this application is to provide an ultra-high-throughput single-cell nucleic acid molecule real-time fluorescence quantitative analysis method, chip and system in view of the above-mentioned deficiencies in the prior art.
  • this application provides an ultra-high-throughput single-cell nucleic acid real-time fluorescence quantitative analysis method, which includes the following steps:
  • a microwell array chip is provided.
  • the microwell array chip is provided with at least one microwell array area, the microwell array area includes a plurality of microwells, and the inner wall of the microwell is modified with at least one DNA probe. needle;
  • the micropore has a size and shape that can only accommodate a single cell in one micropore.
  • the diameter of the micropore is 1-100 ⁇ m, and the shape is a regular hexagon.
  • the number of micropores single microwell array zone comprising not less than 10 6.
  • microwell array regions are provided on the microwell array chip.
  • the step 1) further includes performing hydrophobic modification on the surface of the micropore array chip and hydrophilic modification on the inside of the micropores.
  • the step 3) includes first passing a cell lysate into the microwell array chip to achieve cell lysis in situ, capturing the target nucleic acid molecule by the modified DNA probe in the microwell, and then A buffer solution is passed into the microwell array chip for washing.
  • the step 4) includes: passing qPCR reaction reagents into the microwell array chip, performing PCR amplification, collecting fluorescent signals through a fluorescence imaging module, and finally performing fluorescence analysis through a fluorescence quantitative analysis module, To achieve single-cell gene expression level analysis.
  • the fluorescence signal collected by the fluorescence imaging module includes a process fluorescence image during the plateau phase of each thermal cycle in the PCR amplification reaction and a final fluorescence image after the PCR amplification reaction is completed.
  • the method for performing fluorescence analysis by the fluorescence quantitative analysis module includes the following steps:
  • S1 Preprocess and store the process fluorescence image and final fluorescence image
  • step S2 Establish an image grid template according to the positions of the microwells on the microwell array chip, and register the process fluorescence image and final fluorescence image processed in step S1 with the image grid template respectively to realize the process fluorescence image and final fluorescence image.
  • an ultra-high-throughput single-cell nucleic acid molecule real-time fluorescence quantitative analysis chip comprising a microwell array chip and a microfluidic packaging structure, the microwell array chip is arranged in the microfluidic packaging Within the structure
  • the microwell array chip is provided with at least one microwell array area on its substrate, the microwell array area has a plurality of microwells, and the microwells have a size and shape that can only accommodate a single cell in one microwell And at least one DNA probe is modified on the inner wall of the micropore.
  • the inner surface of the micropore is subjected to a hydrophilic treatment, and the other surfaces of the micropore array chip except the inner surface of the micropore are subjected to a hydrophobic treatment.
  • the microwell array comprising in-line region on the substrate 10, the number of micropores single microwell array zone comprising not less than 10 6.
  • the micropores are through holes and have a regular polygonal structure.
  • the shape of the micropore is a regular hexagon, and the diameter of its circumscribed circle is 1-100 ⁇ m.
  • the microfluidic packaging structure includes a bottom substrate and an upper cover plate, an upper chip groove is opened in the middle of the lower surface of the upper cover plate, and two sides of the upper chip groove are symmetrically provided with inlets communicating with it.
  • a lower chip groove is provided in the middle of the upper surface of the bottom substrate, and one side of the lower chip groove is provided with a secondary runner groove having the same structure as the sample discharge runner groove ;
  • the upper chip groove and the lower chip groove are positioned opposite to form a chip mounting groove for accommodating the microhole array chip.
  • a sampling flow channel is formed between the upper surface of the bottom substrate, and the sampling flow channel and the secondary flow channel groove are positioned opposite to form a sampling flow channel.
  • the upper cover plate is provided with a sample inlet, a total liquid outlet, a buffer inlet and a buffer outlet; the sample inlet runner groove and the sample outlet runner groove are arranged in the upper layer along the width direction On both sides of the lower surface of the cover plate, the sample inlet and the total liquid outlet are arranged on both sides of the upper cover plate in the width direction, and the buffer inlet and the buffer outlet are arranged on the upper layer along the length direction. Both sides of the cover.
  • the sample injection runner groove, the sample discharge runner groove, and the secondary runner groove have the same structure, and are all tree-like branched structure runners, which have a tree root node port and several sub-node ports;
  • the root node port of the injection channel groove is connected to the injection port, and the child node port is connected to the side of the upper chip groove close to the injection port;
  • the sub-node port of the sample outlet channel groove is connected to the side of the upper chip groove close to the total liquid outlet, and the root node port thereof is connected to the total liquid outlet;
  • the sub-node port of the secondary channel groove is communicated with the side of the lower chip groove close to the total liquid outlet, and the tree root node port is connected to the total liquid outlet;
  • the two ends of the lower chip groove along the length direction are respectively communicated with the buffer inlet and the buffer outlet.
  • the number of the sub-node ports of the tree-like branch structure flow channel is the same as the number of the microwell array area on the microwell array chip.
  • the sub-node ports of the tree-shaped bifurcated flow channel are provided with V-shaped flow channels, and the number and positions of the V-shaped flow channels are the same as the micro-hole array area on the micro-hole array chip.
  • the pointed end of the V-shaped guide groove is communicated with the sub-node port, and the other end is communicated with the side of the micropore array area.
  • this application provides an ultra-high-throughput single-cell nucleic acid molecule real-time fluorescence quantitative analysis integrated rapid detection system, including: a microfluidic chip, an automatic sample loading device, a temperature-controlled thermal cycle device, a fluorescence imaging system, and data Storage analysis system;
  • the automatic sample adding device has degrees of freedom in the X-axis, Y-axis, and Z-axis directions, and is used to automatically add samples and reagents into the microfluidic chip; the microfluidic chip is set in the temperature-controlled thermal cycle On the device
  • the temperature-controlled thermal cycling device is used to realize the thermal cycling temperature control in the PCR amplification reaction of the sample in the microfluidic chip;
  • the fluorescence imaging system is used to collect the fluorescence signal of the sample and transmit it to the data storage and analysis system;
  • the data storage and analysis system analyzes the fluorescence signal of the collected sample, identifies the positive sample, and draws a real-time fluorescence quantitative analysis curve of the positive sample.
  • microfluidic chip of not less than 10 6 micropores, said micropores having a size and shape to accommodate only a single microwell cell.
  • the diameter of the micropore is 1-100 ⁇ m, and the shape is a regular hexagon.
  • the temperature-controlled thermal cycle device includes a mounting seat, a temperature-control base slidably arranged on the mounting seat, and a carrier arranged on the temperature-control base for placing the microfluidic chip Table, heating assembly arranged between the stage and temperature control base, heat dissipation assembly arranged on the mounting base, and a first driving mechanism for driving the temperature control base to slide on the mounting base .
  • the first driving mechanism includes a first slide rail provided on the temperature control base, a first sliding block provided on the first slide rail, and a first active drive provided on the mounting seat.
  • the first sliding block is connected to the first belt, and the mounting seat is connected to the first sliding block.
  • the automatic sample adding device includes a sample adding base fixedly connected to the mounting seat, and an X-axis drive mechanism, a Y-axis drive mechanism, a Z-axis drive mechanism, and a sample adding base arranged on the sample adding base.
  • the mechanical arm; the X-axis drive mechanism, the Y-axis drive mechanism, and the Z-axis drive mechanism are used to realize the movement of the sample application mechanical arm along the X-axis, Y-axis, and Z-axis.
  • the X-axis driving mechanism includes an X-axis slide rail, an X-axis driving pulley and an X-axis driven pulley arranged on the sample adding base, and an X-axis driving pulley and an X-axis driven pulley.
  • the X-axis belt between the driven pulleys, the X-axis motor arranged on the sample loading base and drivingly connected with the X-axis driving pulley, and the X-axis motor slidably arranged on the X-axis slide rail and connected with the Sample mounting plate connected with X axis belt;
  • the Y-axis driving mechanism includes a Y-axis slide rail, a Y-axis driving pulley and a Y-axis driven pulley that are arranged on the sample loading mounting plate, and a Y-axis driving pulley and a Y-axis driven pulley that are arranged on the Y-axis driving pulley and the Y-axis driven pulley.
  • the Y-axis belt between the wheels, the Y-axis motor that is arranged on the sample mounting plate and is connected to the Y-axis driving pulley, and is slidably arranged on the Y-axis slide rail and is connected to the Y-axis.
  • Y-axis slider connected by belt;
  • the Z-axis driving mechanism includes a Z-axis motor fixedly connected to the sample loading mounting plate, a Z-axis pulley drivingly connected with the Z-axis motor, a rotating shaft drivingly connected with the Z-axis pulley, and a fixing sleeve.
  • a drive bushing set on the rotating shaft a Z-axis slider fixedly connected to the Y-axis slider, a Z-axis slider slidably arranged on the Z-axis slider and fixedly connected to the sample loading mechanical arm
  • the fluorescence imaging system includes a broad-spectrum light source, a switchable fluorescence spectroscopy device, and an imaging detector
  • the broad-spectrum light source has a plurality of LED light sources
  • the switchable fluorescence spectroscopy device includes a plurality of fluorescent lights that can be switched into the light path.
  • the excitation light emitted by the broad-spectrum light source is reflected by the fluorescence spectroscopy module and then reaches the sample, and the fluorescence generated by the excitation of the sample passes through the fluorescence spectroscopy module and enters the imaging detector to realize fluorescence imaging.
  • the fluorescence imaging system further includes a support frame fixed on the mounting seat, a support plate fixed on the support frame, a second slide rail provided on the support plate, and a slidable A fluorescent switching slide plate arranged on the second slide rail, a second driving pulley and a second driven pulley, a second belt arranged between the second driving pulley and the second driven pulley, and A second motor arranged on the support plate and drivingly connected with the second driving pulley, and the fluorescent switching slide plate is connected with the second belt.
  • the support plate is provided with an imaging hole
  • the mounting seat can be slid below the support plate to transport the microfluidic chip on the mounting seat to the bottom of the imaging hole, so as to pass fluorescence
  • the imaging system performs fluorescence imaging on the sample in the microfluidic chip.
  • a frame is further provided on the support plate, and the imaging detector is arranged on the frame and directly above the imaging hole;
  • the plurality of fluorescence spectroscopy modules are sequentially arranged on the fluorescence switching sliding plate along the sliding direction of the fluorescence switching sliding plate, and the fluorescence spectroscopy modules are transported one at a time to directly above the imaging hole through the fluorescence switching sliding plate. Switching into the optical path; the broad-spectrum light source is arranged on the side of the imaging hole.
  • the data storage analysis system includes an image preprocessing and storage module, a fluorescence image segmentation and positioning module, and a data statistical analysis module;
  • the fluorescence signal collected by the fluorescence imaging system includes the process fluorescence image of each thermal cycle plateau in the PCR amplification reaction and the final fluorescence image after the PCR amplification reaction is completed;
  • the processing method of the data storage analysis system includes the following steps:
  • the image preprocessing and storage module preprocesses the collected process fluorescence images and final fluorescence images, and stores them;
  • the fluorescence image segmentation and positioning module establishes an image grid template according to the positions of the microwells on the microwell array chip, and matches the process fluorescence image and the final fluorescence image processed in step S1 with the image grid template respectively Accurate, realize the positioning of the micro-hole position on the process fluorescence image and the final fluorescence image;
  • the ultra-high-throughput single-cell nucleic acid real-time fluorescence quantitative analysis method of the present application can realize single-cell capture on the order of 100,000 and million, and real-time quantitative PCR analysis of multiple gene loci can be realized through multiple fluorescent labels.
  • the detection greatly improves the detection throughput, and realizes the analysis of single cells instead of group cell analysis;
  • This application first uses the final fluorescence image to determine the location of the microwells of all positive samples, and then uses the process fluorescence image to extract only the fluorescence images of all positive samples at the plateau period of each thermal cycle, which can reduce the calculation of non-specific data and reduce the calculation In order to realize the rapid drawing of ultra-high-throughput real-time fluorescence quantitative PCR curve.
  • the present application can increase the capillary force of the micropores by performing hydrophilic and hydrophobic treatments on the micropore array chip, which facilitates the inhalation of single cells into the micropores and realizes single cell capture;
  • the microfluidic packaging structure of the present application can realize the uniform distribution of samples by designing the flow channel of the multi-manifold tree-like bifurcation structure, and cover the entire microwell array area, realize efficient and uniform sample injection, and can ensure that each end branch The flow is basically the same, and the smooth advancement of the liquid level is realized.
  • the ultra-high-throughput single-cell nucleic acid molecule real-time fluorescence quantitative analysis integrated rapid detection system of the present application integrates a microfluidic chip, an automatic sample adding device, a temperature-controlled thermal cycle device, a fluorescence imaging system, and a data storage analysis system, which can realize samples
  • the automated detection processing can capture nucleic acid amplification and real-time fluorescence quantitative curve analysis of single cells in the order of one hundred thousand and one million.
  • FIG. 1 is a flowchart of the ultra-high-throughput single-cell nucleic acid real-time fluorescence quantitative analysis method of this application;
  • FIG. 2 is a schematic diagram of the decomposition structure of the ultra-high-throughput single-cell nucleic acid molecule real-time fluorescence quantitative analysis chip of the application;
  • FIG. 3 is a schematic diagram of the structure of the lower surface of the upper cover plate of the application.
  • FIG. 4 is a schematic diagram of the structure when the micro-hole array chip is provided on the lower surface of the upper cover plate of the application;
  • FIG. 5 is a schematic diagram of the structure of the secondary runner groove on the upper surface of the bottom substrate of the application.
  • FIG. 6 is a schematic structural diagram of the overall flow channel in the microfluidic packaging structure of this application.
  • FIG. 7 is a schematic structural diagram of the application's ultra-high-throughput single-cell nucleic acid molecule real-time fluorescence quantitative analysis integrated rapid detection system
  • FIG. 8 is a schematic diagram of the structure of the automatic sample adding device of this application.
  • FIG. 9 is a schematic diagram of another view of the structure of the automatic sample adding device of this application.
  • FIG. 10 is a schematic diagram of a partial structure of the Z-axis driving mechanism of this application.
  • FIG. 11 is a schematic diagram of the structure of the temperature-controlled thermal cycle device of this application.
  • Figure 12 is a schematic diagram of the exploded structure of the temperature-controlled thermal cycle device of this application.
  • FIG. 13 is a schematic structural diagram of the fluorescence imaging system of this application.
  • FIG. 14 is a schematic structural diagram of another viewing angle of the fluorescence imaging system of this application.
  • 15 is a schematic diagram of the structure of the fluorescence spectroscopic module of this application.
  • FIG. 16 is a schematic diagram of the optical path of the fluorescence imaging system of this application.
  • 120 Y-axis slide rail; 121—Y-axis driving pulley; 122—Y-axis driven pulley; 123—Y-axis belt; 124—Y-axis motor; 125—Y-axis slider;
  • 3 Fluorescence imaging system
  • 30 Wide spectrum light source
  • 31 Switchable fluorescence spectroscopy device
  • 32 Imaging detector
  • 33 Secondary frame
  • 34 Second slide rail
  • 35 Fluorescence switching slide
  • 36 the second driving pulley
  • 38 the second driven pulley
  • 39 the second belt
  • 300 the first LED light source
  • 301 the second LED light source
  • 302 the third LED light source
  • 303 the first two directions
  • Color mirror 304—second dichroic mirror
  • 305 collimating lens
  • 310 fluorescence spectroscopic module
  • 311 laens mounting block
  • 312 excitation filter
  • 320 condenser lens
  • 330 support plate
  • 331 imagingg hole
  • 332 Rasteretack
  • the ultra-high-throughput real-time fluorescence quantitative analysis method of single-cell nucleic acid of this embodiment includes the following steps:
  • a microwell array chip is provided.
  • the microwell array chip is provided with at least one microwell array area, the microwell array area includes a plurality of microwells, and at least one DNA probe is modified on the inner wall of the microwell;
  • the micropore has a size and shape that can only accommodate a single cell in one micropore.
  • the diameter of the micropore is 1-100 ⁇ m, and the shape is a regular hexagon.
  • the number of pores include a single microwell array area is not less than 106, a single microwell array region on the order of one hundred thousand to achieve single cell capture.
  • the microwell array chip is provided with 10 in-line microwell array areas, so that the microwell array chip can achieve a million-level ultra-high-throughput single cell capture.
  • DNA probes are used to capture target nucleic acid molecules, and detection of multiple gene loci can be achieved by modifying multiple DNA probes.
  • the substrate of the microporous array chip can be made of materials such as polymer, Si, etc., and a microporous structure with a diameter of 1 ⁇ m-100 ⁇ m can be formed by etching.
  • the surface of the micropore array chip is hydrophobically modified, and the inside of the micropore is modified to be hydrophilic, thereby increasing the capillary force of the micropore and reducing surface liquid residue.
  • step 3) includes first passing the cell lysate into the microwell array chip to achieve cell lysis in situ, capturing the target nucleic acid molecule through the modified DNA probe in the microwell, and then passing it into the microwell array chip Buffer cleaning.
  • step 4) includes: passing qPCR reaction reagents into the microwell array chip, then sealing the microwells, performing PCR amplification, and collecting fluorescent signals through the fluorescence imaging module, and finally performing fluorescence analysis through the fluorescence quantitative analysis module to Realize single-cell gene expression level analysis.
  • the micropores can be sealed by oil seals to realize the isolation of each channel; then enter the PCR reaction device for PCR amplification and fluorescence detection through the fluorescence imaging module.
  • the microwell array chip is provided with a sample inlet, a sample outlet, a buffer inlet and a buffer outlet.
  • the sample to be tested and the qPCR reaction reagents enter through the sample inlet, and the buffer inlet and the buffer outlet are for the entry and exit of reagents such as cell lysate and buffer.
  • the fluorescence signal collected by the fluorescence imaging module includes the process fluorescence image of each thermal cycle plateau in the PCR amplification reaction and the final fluorescence image after the PCR amplification reaction is completed. That is, for each area of the microwell array chip, in the PCR amplification reaction, after the thermal cycle is started, fluorescence images are collected during the low-temperature annealing plateau period of each thermal cycle, which is the process fluorescence image. Then after the entire PCR amplification reaction is completed, the fluorescence image is collected as the final fluorescence image.
  • the final fluorescence image is used to determine the location of the microwells of all positive samples, and then the fluorescence images of all positive samples at the plateau phase of each thermal cycle are extracted through the process fluorescence image to achieve real-time fluorescence quantitative PCR curve drawing.
  • the method for fluorescence analysis performed by the fluorescence quantitative analysis module includes the following steps:
  • S1 Preprocess and store the process fluorescence image and final fluorescence image
  • step S2 Establish an image grid template according to the position of the microwells on the microwell array chip, call the process fluorescence image and the final fluorescence image processed in step S1 to register with the image grid template respectively, to realize the process fluorescence image and the final fluorescence image on the micro Respective positioning of the hole position, corresponding to the position of the process fluorescence image and the final fluorescence image, and can obtain the position information of each micro-hole;
  • the final fluorescence image is used to determine the position of the microwells of all positive samples, and then the fluorescence image is used to extract only the fluorescence images of all the positive samples during each thermal cycle plateau, which can reduce the calculation of non-specific data and reduce the calculation In order to realize the rapid drawing of ultra-high-throughput real-time fluorescent quantitative PCR curve.
  • the DNA probe is used to capture the target nucleic acid molecule, and the detection of multiple gene loci can be achieved by modifying multiple fluorescently labeled multiple DNA probes in the micropore; correspondingly,
  • the fluorescence imaging module is expanded to have the function of imaging a variety of fluorescence, and then the fluorescence quantitative analysis module is used to analyze a variety of fluorescence signals to realize the detection of multiple gene loci.
  • the ultra-high-throughput single-cell nucleic acid molecule real-time fluorescence quantitative analysis chip of this embodiment includes a microwell array chip 5 and a microfluidic packaging structure 6.
  • the microwell array chip 5 is set on the microwell Inside the flow control packaging structure 6;
  • the microwell array chip 5 is provided with at least one microwell array area 50 on its substrate.
  • the microwell array area 50 has a plurality of microwells.
  • the microwells have a size and shape that can only accommodate a single cell in one microwell.
  • At least one DNA probe is modified on the inner wall of the well.
  • the substrate of the microporous array chip 5 can be made of materials such as polymer, Si, etc., and a microporous structure with a diameter of 1 ⁇ m-100 ⁇ m can be formed by etching. And the micro-holes are through holes and have a regular polygonal structure.
  • a single micropore can only accommodate a single cell.
  • the shape of the micropore is a regular hexagon, and the diameter of its circumscribed circle is 1-100 ⁇ m. And comprises a number of pores within a single microwell array region 50 is not less than 106, a single microwell array region 50 on the order of one hundred thousand to achieve single cell capture.
  • the microwell array chip 5 is provided with 10 inline microwell array regions 50, so that the microwell array chip 5 can achieve a total of one million ultra-high-throughput single cell capture.
  • the number of microwells in a single microwell array area 50 and the number of microwell array areas 50 on the microwell array chip 5 can be expanded, which can be expanded to achieve tens of millions of ultra-high-throughput single cell capture.
  • the DNA probe is used to capture the target nucleic acid molecule, and the detection of multiple gene loci can be realized by modifying multiple DNA probes.
  • the inner surface of the micropore is subjected to hydrophilic treatment
  • the other surfaces of the micropore array chip 5 except for the inner surface of the micropore are subjected to hydrophobic treatment.
  • Hydrophilic treatment of the inner surface of the micropores can increase the capillary force of the micropores and facilitate the inhalation of single cells into the micropores; the hydrophobic treatment of the surface can reduce surface liquid residues.
  • the processed micropore array chip 5 can suck the sample through the capillary force of the micropores to realize single cell capture and in-situ observation.
  • the hydrophilic and hydrophobic treatment methods of the microwell array chip 5 are:
  • Hydrophilic treatment immerse the entire microwell array chip 5 in a hydrophilic reagent to react for a certain period of time.
  • the hydrophilic reagent can be at least one of hydrogen peroxide, ammonia, acetic acid, concentrated sulfuric acid, hydrochloric acid or sodium hydroxide;
  • fillers to isolate the inner walls of the micropores;
  • the fillers can be water, trichloroethylene, n-hexane, silicone oil, liquid paraffin, solid paraffin, fluorinated oil, blue At least one of membrane or PDMS;
  • Hydrophobic treatment immerse the filled microwell array chip 5 in a hydrophobic reagent and react for a certain period of time.
  • the hydrophobizing reagent can be perfluorodecyltrimethoxysilane, octadecyltrichlorosilane, or octadecyltrimethyl At least one of oxysilane, hexadecyltriethoxysilane, hexyltriethoxysilane or octyltrichlorosilane;
  • the microwell array chip 5 is ultrasonically cleaned with a cleaning reagent, the fillings in the microwells are removed, and then dried, to obtain the processed microwell array chip 5.
  • the cleaning reagent can be trichloroethylene, acetone, ethanol or isopropanol.
  • This application mainly provides an ultra-high-throughput chip for real-time fluorescence quantitative analysis of single-cell nucleic acid with ultra-high throughput.
  • the main steps of using the chip of this application to perform fluorescence quantitative analysis are as follows:
  • the ultra-high-throughput single-cell capture of the million-level can be achieved by the chip with micro-pores of the present application, so as to provide chip support for real-time fluorescence quantitative analysis method and system of single-cell nucleic acid with ultra-high-throughput.
  • the microfluidic package structure 6 includes a bottom substrate 60 and an upper cover 61.
  • the upper cover 61 is provided with an upper chip groove 62 in the middle of the lower surface, and two sides of the upper chip groove 62 are symmetrically provided with communicating with it.
  • a lower chip groove 66 is provided in the middle of the upper surface of the bottom substrate 60, and one side of the lower chip groove 66 is provided with a secondary runner having the same structure as the sample discharge runner groove 64 Slot 65;
  • the upper chip groove 62 and the lower chip groove 66 are positioned opposite to form a chip mounting groove for accommodating the microhole array chip 5, the sample flow channel 63 and the bottom substrate 60 A sampling flow channel is formed between the upper surfaces, and the sampling flow channel groove 64 and the secondary flow channel groove 65 are positioned directly opposite to form a sampling flow channel.
  • microfluidic packaging structure 6 is used to encapsulate the microporous array chip 5, and through the design of the flow channel, rapid sample injection and uniform sample distribution can be realized.
  • the upper cover 61 is provided with a sample inlet 67, a total liquid outlet 68, a buffer inlet 69, and a buffer outlet 70; the sample inlet runner groove 63 and the sample outlet runner groove 64 are arranged on the upper cover along the width direction.
  • the sample inlet 67 and the total liquid outlet 68 are arranged on both sides of the upper cover 61 in the width direction, and the buffer inlet 69 and the buffer outlet 70 are arranged on the upper cover 61 along the length direction. On both sides.
  • the sample injection runner groove 63, the sample discharge runner groove 64 and the secondary runner groove 65 have the same structure, and are all tree-like branched structure runners, which have a tree root node port 71 and several sub-node ports 72;
  • the root node port 71 of the injection channel groove 63 is connected to the injection port 67, and the child node port 72 is connected to the side of the upper chip groove 62 close to the injection port 67;
  • the sub-node port 72 of the sample outlet channel groove 64 is connected to the side of the upper chip groove 62 close to the total liquid outlet 68, and the root node port 71 is connected to the total liquid outlet 68;
  • the sub-node port 72 of the secondary channel groove 65 is communicated with the side of the lower chip groove 66 close to the total liquid outlet 68, and its root node port 71 is connected to the total liquid outlet 68;
  • the two ends of the lower chip slot 66 along the length direction are respectively connected with the buffer inlet 69 and the buffer outlet 70.
  • the structure of the overall flow channel in the microfluidic packaging structure 6 is shown in FIG. 6.
  • the sample Under the action of the driving pump, the sample enters through the injection port 67, and is evenly and quickly distributed to the upper surface of the micropore array chip 5 through the injection channel 63.
  • the sample is sucked into the micropore by the capillary force of the micropore, thereby realizing the micropore
  • the excess sample is discharged from the sample outlet channel 64 on the other side of the microwell array chip 5 through the total liquid outlet 68.
  • the buffer enters the lower chip groove 66 through the buffer inlet 69 and reaches the lower surface of the microwell array chip 5, and exchanges substances from the bottom of the microwell with the inside of the microwell, and excess buffer can be discharged from the buffer outlet 70.
  • the sample flowing into the micropore on the upper surface of the micropore array chip 5 needs to overcome the capillary force to flow out from the bottom of the micropore and flow into the lower area.
  • the sample flow rate of the driving pump is not enough to make the sample It can overcome the capillary force of the micropores, so the sample will not flow out from the lower part of the micropores to ensure that the cells can be fixed in the micropores.
  • the upper and lower regions of the micropore array chip 5 can be stratified, and the upper and lower layers can be independent Control, can pass in different reagents.
  • the total liquid outlet 68 is connected to a vacuum pump, and the cleaning liquid enters from the injection port 67 and is distributed to the upper surface of the microwell array chip 5 through the injection channel groove 63.
  • the vacuum pump The suction effect enables the cleaning liquid to overcome the capillary force of the micropores, and the liquid in the micropores can flow out from the bottom of the micropores into the secondary channel groove 65, and then be discharged from the total liquid outlet 68.
  • the flow channel with a tree-like bifurcation structure can achieve uniform distribution of samples and cover the entire microwell array area 50, so that each microwell can be sampled, and the flow rate of each end branch can be basically the same , Realize the smooth advancement of the liquid level.
  • the number of the sub-node ports 72 of the flow channel of the tree-like bifurcation structure is the same as the number of the microwell array area 50 on the microwell array chip 5, and both are 10.
  • the sub-node port 72 of the flow channel of the tree-like bifurcation structure is provided with V-shaped diversion grooves 73, and the number and positions of the V-shaped diversion grooves 73 correspond one-to-one with the microwell array area 50 on the microwell array chip 5.
  • each V-shaped diversion groove 73 corresponds to a side of the microwell array area 50; the pointed end of the V-shaped diversion groove 73 is connected to the sub-node port 72, and the other end is connected to the microwell array area.
  • the side of 50 is connected.
  • the arrangement of the V-shaped guide groove 73 can facilitate the rapid and uniform flow of the liquid, as well as the distribution and collection.
  • the V-shaped guide groove 73 of the sub-node port 72 facilitates the rapid and uniform introduction of the incoming sample above the microwell array area 50.
  • the V-shaped diversion groove 73 of the sub-node port 72 facilitates the rapid collection of the liquid in the chip mounting slot into the sub-node port 72 for efficient discharge.
  • an ultra-high-throughput single-cell nucleic acid molecule real-time fluorescent quantitative analysis integrated rapid detection system of this embodiment includes: a microfluidic chip 4, an automatic sample adding device 1, a temperature-controlled thermal cycle device 2 , Fluorescence imaging system 3 and data storage and analysis system;
  • the automatic sample adding device 1 has degrees of freedom in the X-axis, Y-axis and Z-axis directions, and is used to automatically add samples and reagents into the microfluidic chip 4; the microfluidic chip 4 is set on the temperature-controlled thermal cycle device 2;
  • the temperature-controlled thermal cycle device 2 is used to realize the thermal cycle temperature control of the sample in the microfluidic chip 4 in the PCR amplification reaction;
  • the fluorescence imaging system 3 is used to collect the fluorescence signal of the sample and transmit it to the data storage and analysis system;
  • the data storage analysis system analyzes the fluorescence signals of the collected samples, identifies the positive samples, and draws the real-time fluorescence quantitative analysis curve of the positive samples.
  • the data storage and analysis system can be embedded in a host computer (such as a computer), and the host computer is communicatively connected with the fluorescence imaging system 3 and can accept the data collected by the fluorescence imaging system 3.
  • a is not less than 106 pores 4 on the microfluidic chip
  • the microwell array is provided, in micropores having a pore size and shape can only accommodate a single cell.
  • the microfluidic chip 4 is provided on the control microwell array region 10 has inline, each microwell array is provided with not less than the micropores 106, the entire microfluidic chip 7 is provided with pores less than 10 4, so that the microwell array chip to reach a total of the order of one million ultra high throughput single cell capture.
  • at least one DNA probe is modified on the inner wall of the micropore, and the DNA probe is used to capture the target nucleic acid molecule, and the detection of multiple gene loci can be achieved by modifying multiple DNA probes.
  • the diameter of the micropores is 1-100 ⁇ m, and the shape is a regular hexagon.
  • the substrate of the microporous array chip can be made of materials such as polymer, Si, etc., and a microporous structure with a diameter of 1 ⁇ m-100 ⁇ m can be formed by etching.
  • the microfluidic chip 4 is provided with a conventional sample inlet, a sample outlet, a buffer inlet and a buffer outlet. The sample to be tested and the qPCR reaction reagents enter through the sample inlet, and the buffer inlet and the buffer outlet are for the entry and exit of reagents such as cell lysate and buffer.
  • the samples in the microfluidic chip 4 are amplified by PCR on the temperature-controlled thermal cycler 2, and the fluorescence signal is collected by the fluorescence imaging system 3.
  • the main working process is: put the microfluidic chip 4 into the temperature-controlled thermal cycler 2, and then add relevant reagents to the microfluidic chip 4 through the automatic sample adding device 1, which mainly includes: first pass the cell lysate to achieve Cells are lysed in situ, the target nucleic acid molecules are captured by the modified DNA probes in the micropores, and then washed with buffer; finally, the qPCR reagents are introduced, and then the micropores are sealed, PCR amplification is carried out, and collected by the fluorescence imaging system 3 The fluorescence signal is finally analyzed by the data storage analysis system to realize the single-cell gene expression level analysis.
  • oil seals can be used to seal the micropores to achieve isolation of each channel; then PCR amplification and fluorescence detection are performed.
  • the sample adding scheme of the automatic sample adding device 1, the temperature control scheme of the temperature-controlled thermal cycle device 2, the imaging scheme of the fluorescence imaging system 3, and the analysis processing scheme of the data storage analysis system can be implemented by the following specific embodiments.
  • the fluorescence signal of the fluorescence imaging module system includes the process fluorescence image of each thermal cycle plateau in the PCR amplification reaction and the final fluorescence image after the PCR amplification reaction is completed. That is, for each area of the microfluidic chip 4, in the PCR amplification reaction, after the thermal cycle is started, fluorescent images are collected during the low-temperature annealing plateau of each thermal cycle, which is the process fluorescence image. Then after the entire PCR amplification reaction is completed, the fluorescence image is collected as the final fluorescence image.
  • the final fluorescence image is used to determine the location of the microwells of all positive samples, and then the fluorescence images of all positive samples at the plateau phase of each thermal cycle are extracted through the process fluorescence image to achieve real-time fluorescence quantitative PCR curve drawing.
  • the analysis and processing of the data storage and analysis system can be realized by the following scheme: the data storage and analysis system includes an image preprocessing and storage module, a fluorescence image segmentation and positioning module, and a data statistical analysis module; the fluorescence imaging system 3 collects The fluorescence signal includes the process fluorescence image of each thermal cycle plateau in the PCR amplification reaction and the final fluorescence image after the PCR amplification reaction is completed.
  • the processing method of the data storage analysis system includes the following steps:
  • the image preprocessing and storage module preprocesses the collected process fluorescence images and final fluorescence images, and stores them;
  • the fluorescence image segmentation and positioning module establishes an image grid template according to the position of the microwells on the microwell array chip, and registers the process fluorescence image and final fluorescence image processed in step S1 with the image grid template respectively to realize the process fluorescence image And the positioning of the micro-hole position on the final fluorescence image;
  • the final fluorescence image is used to determine the location of the microwells of all positive samples, and then the fluorescence image is used to extract only the fluorescence images of all positive samples during each thermal cycle plateau, which can reduce the calculation of non-specific data and reduce the calculation In order to realize the rapid drawing of ultra-high-throughput real-time fluorescent quantitative PCR curve.
  • the temperature-controlled thermal cycle device 2 includes a mounting base 20, a temperature-controlling base 21 slidably disposed on the mounting base 20, and a temperature-controlling base 21.
  • the microfluidic chip 4 is placed on the stage 22 and covered and sealed by a transparent cover.
  • a heat conductor 24 is provided on the temperature control base 21, and the heat conductor 24 has a plurality of heat conduction fins to facilitate rapid heat dissipation.
  • a copper plate 27 is arranged on the heat exchanger 24, the heating device is a Peltier or a thermocouple, and is arranged on the copper plate 27, the carrier 22 is arranged on the heating device, and the microfluidic chip 4 on the carrier 22 is controlled by the heating device. The sample is heated.
  • the heat dissipation assembly 25 is a plurality of heat dissipation fans arranged on the mounting base 20, and heat dissipation is accelerated by the heat dissipation fans.
  • the first driving mechanism 26 includes a first sliding rail 260 arranged on the temperature control base 21, a first sliding block 261 arranged on the first sliding rail 260, a first driving pulley 262 arranged on the mounting seat 20, and The first driven pulley 263, the first belt 264 disposed between the first driving pulley 262 and the first driven pulley 263, and the first motor 265 drivingly connected with the first driving pulley 262;
  • the block 261 is connected with the first belt 264, and the mounting base 20 is connected with the first sliding block 261.
  • the first motor 265 drives the temperature control base 21 to slide along the X axis, so that the temperature control base 21 is transported to the top of the heat dissipation fan or to the discrete hot fan.
  • the temperature control base 21 is in the initial position, which is located on the left side of the fluorescence imaging system 3.
  • the temperature control base 21 is transported to the reaction position by the first driving mechanism 26, that is, below the fluorescence imaging system 3 and above the heat dissipation wind.
  • the heating device heats the sample; when cooling is required, the cooling fan below is activated to quickly cool down.
  • the automatic sample adding device 1 includes a sample adding base 10 fixedly connected to the mounting base 20 and an X-axis driving mechanism arranged on the sample adding base 10 11.
  • the Y-axis drive mechanism 12, the Z-axis drive mechanism 13, and the sample application mechanical arm 14; the X-axis drive mechanism 11, the Y-axis drive mechanism 12, and the Z-axis drive mechanism 13 are used to realize the sample application mechanical arm 14 along the X axis, Movement in the Y-axis and Z-axis directions.
  • a sample adding needle 140 is provided on the sample adding mechanical arm 14, which can suck samples and reagents and add them to the microfluidic chip 4.
  • the X-axis driving mechanism 11 includes an X-axis slide rail 110, an X-axis driving pulley 111 and an X-axis driven pulley 112 arranged on the sample adding base 10, and an X-axis driving pulley 111 and an X-axis driving pulley.
  • the motor drives the sample mounting plate 115 to move along the X-axis direction through the X-axis driving pulley 111, the X-axis driven pulley 112, and the X-axis belt 113, thereby driving the Y-axis driving mechanism 12 and the Z-axis driving mechanism 13 ,
  • the sample adding mechanical arm 14 moves along the X axis as a whole.
  • the Y-axis driving mechanism 12 includes a Y-axis slide rail 120 arranged on the sample mounting plate 115, a Y-axis driving pulley 121 and a Y-axis driven pulley 122, and a Y-axis driving pulley 121 and Y
  • Y-axis slider 125 connected to the Y-axis belt 123.
  • the Y-axis motor 124 drives the Y-axis slider 125 to move in the Y-axis direction through the Y-axis driving pulley 121, the Y-axis driven pulley 122, and the Y-axis belt 123, thereby driving the sample application mechanical arm 14 to move in the Y-axis direction.
  • the Z-axis driving mechanism 13 includes a Z-axis motor 130 fixedly connected to the sample mounting plate 115, a Z-axis pulley 131 drivingly connected to the Z-axis motor 130, and a rotating shaft 132 drivingly connected to the Z-axis pulley 131.
  • the drive shaft sleeve 133 fixedly sleeved on the rotating shaft 132, the Z-axis slider 134 fixedly connected to the Y-axis slider 125, and the Z-axis slider 134 slidably arranged on the Z-axis slider 134 and fixedly connected to the sample loading mechanical arm 14
  • the Z-axis slide rail 135 and the drive bar 136 fixed to the sample loading robot arm 14 for cooperating with the drive sleeve 133 drive the drive bar 136 to move in the Z-axis direction through the rotation of the drive sleeve 133, and the Y-axis slider
  • a shaft hole for the rotating shaft 132 to pass through is opened on the 125.
  • the Z-axis pulley 131 includes a driving pulley drivingly connected with the Z-axis motor 130 and a driven pulley connected with the main pulley through a belt.
  • the sample mounting plate 115 is provided with a rotating shaft seat 137 for installing the rotating shaft 132.
  • the Z-axis motor 130 drives the rotating shaft 132 to rotate through the Z-axis pulley 131, and the drive sleeve 133 on the rotating shaft 132 rotates to drive the drive bar 136 to move in the Z-axis direction, so as to realize the movement of the sample loading robot 14 in the Z-axis direction.
  • the drive shaft sleeve 133 and the drive bar 136 can adopt friction drive cooperation (for example, the drive shaft sleeve 133 is a friction sleeve or a friction wheel, and the drive bar 136 is a friction plate or a rack plate or a friction surface. Through friction, The Z-direction movement of the drive bar 136 is driven by the rotation of the drive shaft sleeve 133.
  • Gear meshing can also be used (for example, the drive shaft sleeve 133 is a gear sleeve with teeth provided on the outer circumference, and the drive bar 136 is a rack, which passes through the teeth on the gear sleeve.
  • the part meshes with the rack, and converts the rotation of the drive shaft sleeve 133 into the Z-direction movement of the drive bar 136).
  • the drive shaft sleeve 133 is a friction sleeve
  • the drive bar 136 is a rack plate. The rotation of the friction sleeve is converted into a Z-direction movement of the rack plate through the friction between the friction sleeve and the rack plate.
  • the X-axis drive mechanism 11, the Y-axis drive mechanism 12, and the Z-axis drive mechanism 13 can realize the movement of the sample application mechanical arm 14 along the X-axis, Y-axis, and Z-axis directions, so as to realize the sample, Automatic addition of various reagents.
  • the fluorescence imaging system 3 includes a broad-spectrum light source 30, a switchable fluorescence spectroscopic device 31 and an imaging detector 32, a broad-spectrum light source 30 and multiple LED light sources,
  • the switchable fluorescence spectroscopic device 31 includes a plurality of fluorescence spectroscopic modules 310 that can be switched into the optical path;
  • the excitation light emitted by the broad-spectrum light source 30 is reflected by the fluorescence spectroscopic module 310 and reaches the sample.
  • the fluorescence generated by the sample is transmitted through the fluorescence spectroscopic module 310 and then enters the imaging detector 32 to realize fluorescence imaging.
  • the broad spectrum light source 30 also includes a light splitting path to transmit multiple LED light sources to the fluorescent light splitting module 310.
  • the fluorescence imaging system 3 further includes a support frame 33 fixed on the mounting base 20, a support plate 330 fixed on the support frame 33, a second slide rail 34 arranged on the support plate 330, and Slide the fluorescent switching slide plate 35 arranged on the second slide rail 34, the second driving pulley 37 and the second driven pulley 38, and the second driving pulley 37 and the second driven pulley 38 arranged between the second driving pulley 37 and the second driven pulley 38.
  • Two belts 39 and a second motor 36 arranged on the supporting plate 330 and drivingly connected to the second driving pulley 37, and the fluorescent switching slide plate 35 is connected to the second belt 39.
  • the supporting plate 330 is provided with an imaging hole 331, and the mounting base 20 can be slid below the supporting plate 330 to transport the microfluidic chip 4 on the mounting base 20 below the imaging hole 331, so that the microfluidic imaging system 3 is used to pair the micro The sample in the fluid control chip 4 undergoes fluorescence imaging.
  • a frame 332 is also provided on the support plate 330, and the imaging detector 32 is arranged on the frame 332 and is directly above the imaging hole 331;
  • a plurality of fluorescence spectroscopy modules 310 are sequentially arranged on the fluorescence switching slide 35 along the sliding direction of the fluorescence switching slide 35, and one fluorescence spectroscopy module 310 is transported to directly above the imaging hole 331 through the fluorescence switching slide 35 at a time to switch into the optical path;
  • the light source 30 is provided on the side of the imaging hole 331.
  • the wide-spectrum light source has 303 LED light sources with different wavelengths to achieve full-band visible light (wavelength 400nm-700nm) coverage.
  • the light splitting path includes three collimating lenses 305 arranged at the exit ends of the three LED light sources and a number of dichroic mirrors for reflecting or transmitting the light emitted by the LED light sources to the fluorescent light splitting module 310.
  • the three LED light sources are a first LED light source 300, a second LED light source 301, and a third LED light source 302, and the dichroic mirror includes two: a first dichroic mirror 303 and a second LED light source. To the color mirror 304.
  • the light emitted by the first LED light source 300 is collimated by the collimator lens 305 and then transmitted through the first dichroic mirror 303 and the second dichroic mirror 304 in sequence, and enters the fluorescence spectroscopic module 310; the light emitted by the second LED light source 301 is collimated
  • the collimating lens 305 is collimated and reflected by the first dichroic mirror 303, and then transmitted through the second dichroic mirror 304 to enter the fluorescence beam splitting module 310; the light emitted by the third LED light source 302 is collimated by the collimating lens 305
  • the second dichroic mirror 304 is reflected to the fluorescence spectroscopic module 310.
  • the fluorescence spectroscopy module 310 includes four, and each fluorescence spectroscopy module 310 includes a lens mounting block 311 and an excitation filter 312 arranged therein.
  • the wavelength allowed by each excitation filter 312 is different, and the realization is different. Fluorescence filtering.
  • the lens mounting block 311 has holes all around it. When the fluorescence spectroscopic module 310 switches into the light path, the fluorescence spectroscopic module 310 is above the imaging hole 331 and directly below the imaging detector 32, and the LED light source is at
  • the imaging detector 32 is a high-sensitivity CMOS camera or a CCD camera, and a condenser 320 is also provided in front of the camera.
  • the light emitted by the broad-spectrum light source 30 passes through the light splitting path and is reflected on the sample by the fluorescence splitting module 310.
  • the fluorescence generated by the sample passes through the excitation filter 312 and then reaches the CMOS camera through the condenser 320 for fluorescence imaging.
  • DNA probes are used to capture target nucleic acid molecules.
  • the detection of multiple gene loci can be achieved by modifying multiple fluorescently labeled DNA probes in the micropore; correspondingly, the fluorescence imaging system 3 needs to be expanded to have The function of imaging a variety of fluorescence, and then using the data storage analysis system to analyze a variety of fluorescence signals, you can realize the detection of multiple gene loci. Therefore, in this embodiment, the full-wavelength visible light (wavelength is 400nm-700nm) coverage is achieved by setting three light sources with different wavelengths, and four switchable fluorescence spectroscopic modules 310 are used to filter different fluorescent lights, and finally different fluorescent lights are used. Imaging.
  • the workflow of the ultra-high-throughput single-cell nucleic acid molecule real-time fluorescence quantitative analysis integrated rapid detection system includes:
  • the stage 22 is initialized, the stage 22 is at the initial sample loading position, the microfluidic chip 4 is loaded into the stage 22, the sample is added to the microfluidic chip 4 through the automatic sample adding device 1, and the sample is added to the microfluidic chip 4 through the automatic sample loading device 1.
  • Capillary force generated by the micropore structure on the control chip 4 captures single cells;
  • the entire stage 22 is moved to the reaction zone (that is, the detection zone) by the first driving mechanism 26, the microfluidic chip 4 is moved below the imaging hole 331, the PCR thermal cycle is started, and the heating device heats the sample to the setting Temperature, and collect fluorescence images during the plateau period of each thermal cycle.
  • switch the fluorescence spectroscopy module 310 to collect one by one; then move through the stage 22 to move the next area of the microfluidic chip 4 Just below the imaging hole 331, perform fluorescence imaging of the next area; stitch the collected images to form a fluorescence image including the entire sample area of the microfluidic chip 4, which is the process fluorescence image of the thermal cycle plateau period .
  • the fluorescence image of the entire sample area of the microfluidic chip 4 is collected, that is, the final fluorescence image;
  • the data storage analysis system calls the collected fluorescence signals and analyzes them to obtain real-time fluorescence quantitative PCR curves.
  • the data storage analysis system includes image preprocessing and storage modules, fluorescence image segmentation and positioning modules, and data statistical analysis modules.
  • the specific analysis is Methods include:
  • the image preprocessing and storage module preprocesses the collected process fluorescence image and final fluorescence image (which may include image stitching, noise reduction filtering, image enhancement, etc.), and stores;
  • the fluorescence image segmentation and positioning module establishes an image grid template according to the position of the microwells on the microwell array chip, and registers the process fluorescence image and final fluorescence image processed in step S1 with the image grid template respectively to achieve Positioning of the micro-hole position on the process fluorescence image and the final fluorescence image;
  • the above-mentioned process is a fluorescence signal processing for one type of fluorescence, and when multiple types of fluorescence signals are included, the above-mentioned steps can be processed separately.

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Abstract

一种超高通量的单细胞核酸实时荧光定量分析方法及使用该方法的超高通量单细胞核酸分子实时荧光定量分析芯片和系统。该方法包括以下步骤：1）提供一种微孔阵列芯片(5)，微孔阵列芯片(5)上设置有至少一个微孔阵列区(50)，微孔阵列区(50)包括多个微孔，微孔内壁上修饰有至少一个DNA探针；2）将待测样品加入微孔阵列芯片(5)中，通过微孔捕获单细胞；3）通过DNA探针捕获目标核酸分子；4）进行PCR扩增检测，通过荧光定量分析，实现单细胞基因表达水平分析。该方法、芯片和系统可以实现十万量级、百万量级的单细胞捕获，通过多种荧光标记可实现多个基因位点的实时定量PCR分析检测，相比于现有产品，极大的提升了检测通量，并且实现了单个细胞而非群体细胞的分析。

Description

超高通量单细胞核酸分子实时荧光定量分析方法、芯片和系统
交叉引用
本申请同时要求在2019年9月25日提交中国专利局、申请号为201910912751.3、申请名称为“超高通量的单细胞核酸实时荧光定量分析方法”，2019年9月25日提交中国专利局、申请号为201910912693.4、申请名称为“超高通量单细胞核酸分子实时荧光定量分析芯片”，2019年9月25日提交中国专利局、申请号为201910911821.3、申请名称为“超高通量单细胞核酸分子实时荧光定量分析一体化快速检测系统”三件中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本申请涉及基因检测技术领域，特别涉及一种超高通量单细胞核酸分子实时荧光定量分析方法、芯片和系统。
背景技术
机体的生长、发育、分化、衰老和病理病变等均与基因的差异表达有关。肿瘤的发生发展及转移也与基因的突变和差异表达有关，肿瘤组织中心的细胞、周围的细胞、转移灶的细胞等，也因基因组和转录表达谱的差异，导致功能特性的不同，影响和决定肿瘤的治疗等结果。
传统的基因表达研究方法通常在mRNA水平来衡量某个基因的表达。对于mRNA水平的表达通常用实时荧光定量PCR(real-time PCR,RT-PCR)来实现。当前的荧光定量RT-PCR(RT-qPCR)，只能在细胞群体水平观察多细胞平均的结果。在细胞群体水平上进行的，最终得到的结果，其实是多个细胞的平均，往往丢失了细胞异质性的信息及单细胞功能多样性的关键信息。
随着单细胞分析技术发展，单细胞多基因检测系统应运而生。其通常是利用微流控通道制作独立的单元，将单细胞单独隔离在独立单元中，并进行cDNA扩增，每次最多捕获几百至几千个单细胞，然后再配合仪器进行核酸扩增检测，但现有的微流控芯片的微孔通道数量一般仅为几百至几万，每次捕获的单细胞数量少，检测通量低，无法满足超高通量细胞核酸分子检测的需求。
因此当前单细胞分析方法存在通量低等缺陷，且还需结合再结合核酸荧光实时定量分析仪器。而现有核酸实时定量分析仪器是基于群体细胞核酸分子分析，丢失了细胞异质性的信息及单细胞功能多样性的关键信息。所以现在需要提供更可靠的方案。
申请内容
本申请所要解决的技术问题在于针对上述现有技术中的不足，提供一种超高通量单细胞核酸分子实时荧光定量分析方法、芯片和系统。
为解决上述技术问题，一方面，本申请提供一种超高通量的单细胞核酸实时荧光定量分析方法，包括以下步骤：
1)提供一种微孔阵列芯片，所述微孔阵列芯片上设置有至少一个微孔阵列区，所述微孔阵列区包括多个微孔，所述微孔内壁上修饰有至少一个DNA探针；
2)将待测样品加入所述微孔阵列芯片中，通过所述微孔捕获单细胞；
3)通过所述DNA探针捕获目标核酸分子；
4)进行PCR扩增检测，通过荧光定量分析，实现单细胞基因表达水平分析。
可选地，所述微孔具有在一个微孔中只能容纳单个细胞的尺寸和形状。
可选地，所述微孔的直径为1-100μm，形状为正六边形。
可选地，单个所述微孔阵列区内包括的微孔数量不小于10 6个。
可选地，所述微孔阵列芯片上设置有10个所述微孔阵列区。
可选地，所述步骤1)中还包括对所述微孔阵列芯片的表面进行疏水修饰，对所述微孔内部进行亲水修饰。
可选地，所述步骤3)中包括先向所述微孔阵列芯片中通入细胞裂解液，实现细胞原位裂解，通过所述微孔内修饰的DNA探针捕获目标核酸分子，然后再向所述微孔阵列芯片中通入缓冲液清洗。
可选地，所述步骤4)中包括：向所述微孔阵列芯片中通入qPCR反应试剂，进行PCR扩增，并通过荧光成像模块采集荧光信号，最后通过荧光定量分析模块进行荧光分析，以实现单细胞基因表达水平分析。
可选地，所述荧光成像模块采集的荧光信号包括PCR扩增反应中每次热循环平台期的过程荧光图像以及PCR扩增反应完成后的最终荧光图像。
可选地，所述荧光定量分析模块进行荧光分析的方法包括以下步骤：
S1：对过程荧光图像和最终荧光图像进行预处理，并存储；
S2：根据所述微孔阵列芯片上的微孔位置建立图像网格模板，将所述步骤S1处理后的过程荧光图像、最终荧光图像分别与图像网格模板配准，实现过程荧光图像和最终荧光图像上微孔位置的定位；
S3：针对最终荧光图像，提取阳性信号样本所在的微孔位置信息，作为目标微孔位置；
S4：针对过程荧光图像，提取与所述目标微孔位置对应的阳性样本所在的微孔的荧光强度信息，并绘制阳性样本的实时荧光定量PCR曲线。
另一方面，本申请提供：一种超高通量单细胞核酸分子实时荧光定量分析芯片，包括微孔阵列芯片和微流控封装结构，所述微孔阵列芯片设置在所述微流控封装结构内；
所述微孔阵列芯片在其基底上设置有至少一个微孔阵列区，所述微孔阵列区具有多个微孔，所述微孔具有在一个微孔中只能容纳单个细胞的尺寸和形状，且所述微孔内壁上修饰有至少一个DNA探针。
可选地，所述微孔的内表面经亲水处理，所述微孔阵列芯片除所述微孔内表面的其他表面经疏水处理。
可选地，所述微孔阵列区包括一字排列在基底上的10个，单个所述微孔阵列区内包括的微孔数量不小于10 6个。
可选地，所述微孔为通孔，且为正多边形结构。
可选地，所述微孔形状为正六边形，其外接圆的直径为1-100μm。
可选地，所述微流控封装结构包括底层基板和上层盖板，所述上层盖板的下表面中部开设有上芯片槽，所述上芯片槽的两侧对称设置有与之连通的进样流道槽和出样流道槽；所述底层基板上表面中部设置有下芯片槽，所述下芯片槽的一侧设置有与所述出样流道槽的结构相同的副流道槽；
所述上层盖板与底层基板贴合连接后，所述上芯片槽和下芯片槽位置正对形成用于容纳所述微孔阵列芯片的芯片安装槽，所述进样流道槽和所述底层基板的上表面之间形成进样流道，所述出样流道槽和副流道槽位置正对形成出样流道。
可选地，所述上层盖板上设置有进样口、总出液口、缓冲液入口和缓冲液出口；所述进样流道槽和出样流道槽沿宽度方向设置在所述上层盖板的下表面两侧，所述进样口、总出液口沿宽度方向设置在所述上层盖板的两侧，所述缓冲液入口和缓冲液出口沿长度度方向设置在所述上层盖板的两侧。
可选地，所述进样流道槽、出样流道槽和副流道槽结构相同，均为树状分叉结构流道，其具有一个树根节点端口和若干子节点端口；
所述进样流道槽的树根节点端口与所述进样口连通，其子节点端口连通至所述上芯片槽的靠近所述进样口的一侧；
所述出样流道槽的子节点端口与所述上芯片槽的靠近所述总出液口的一侧连通，其树根节点端口连通至所述总出液口；
所述副流道槽的子节点端口与所述下芯片槽的靠近所述总出液口的一侧连通，其树根节点端口连通至所述总出液口；
所述下芯片槽沿长度方向的两端分别与所述缓冲液入口和缓冲液出口连通。
可选地，所述树状分叉结构流道的子节点端口的数量与所述微孔阵列芯片上的微孔阵列区的个数相同。
可选地，所述树状分叉结构流道的子节点端口设置有V型导流槽，所述V型导流槽的数量和位置与所述微孔阵列芯片上的微孔阵列区一一对应；所述V型导流槽的尖口端与所述子节点端口连通，另一端与所述微孔阵列区的侧部连通。
又一方面，本申请提供一种超高通量单细胞核酸分子实时荧光定量分析一体化快速检测系统，包括：微流控芯片、自动加样装置、温控热循环装置、荧光成像系统以及数据存储分析系统；
所述自动加样装置具有X轴、Y轴和Z轴方向的自由度，用于将样品和试剂自动加入所述微流控芯片内；所述微流控芯片设置在所述温控热循环装置上；
所述温控热循环装置用于实现所述微流控芯片中的样品进行PCR扩增反应中的热循环温度控制；
所述荧光成像系统用于采集样品的荧光信号并传输至所述数据存储分析系统；
所述数据存储分析系统对采集的样品的荧光信号进行分析，识别阳性样本，并绘制出阳性样本的实时荧光定量分析曲线。
可选地，所述微流控芯片上设置有不小于10 6个微孔，所述微孔具有在一个微孔中只能容纳单个细 胞的尺寸和形状。
可选地，所述微孔的直径为1-100μm，形状为正六边形。
可选地，所述温控热循环装置包括安装座、可滑动设置在所述安装座上的温控底座、设置在所述温控底座上的用于放置所述微流控芯片的载物台、设置在所述载物台和温控底座之间的加热组件、设置在所述安装座上的散热组件以及用于驱动所述温控底座在所述安装座上滑动的第一驱动机构。
可选地，所述第一驱动机构包括设置在所述温控底座上的第一滑轨、设置在所述第一滑轨上的第一滑块、设置在所述安装座上第一主动带轮和第一从动带轮、设置在所述第一主动带轮和第一从动带轮之间的第一皮带以及与所述第一主动带轮驱动连接的第一电机；所述第一滑块与所述第一皮带连接，所述安装座连接在所述第一滑块上。
可选地，所述自动加样装置包括固接在所述安装座上的加样底座以及设置在所述加样底座上的X轴驱动机构、Y轴驱动机构、Z轴驱动机构、加样机械臂；所述的X轴驱动机构、Y轴驱动机构、Z轴驱动机构用于实现所述加样机械臂沿X轴、Y轴和Z轴方向的运动。
可选地，所述X轴驱动机构包括设置在所述加样底座上的X轴滑轨、X轴主动带轮和X轴从动带轮、设置在所述X轴主动带轮和X轴从动带轮之间的X轴皮带、设置在所述加样底座上且与所述X轴主动带轮驱动连接的X轴电机以及可滑动设置在所述X轴滑轨上且与所述X轴皮带连接的加样安装板；
所述Y轴驱动机构包括设置在所述加样安装板上的Y轴滑轨、Y轴主动带轮和Y轴从动带轮、设置在所述Y轴主动带轮和Y轴从动带轮之间的Y轴皮带、设置在所述加样安装板上且与所述Y轴主动带轮驱动连接的Y轴电机以及可滑动设置在所述Y轴滑轨上且与所述Y轴皮带连接的Y轴滑块；
所述Z轴驱动机构包括固接在所述加样安装板上的Z轴电机、与所述Z轴电机驱动连接的Z轴带轮、与所述Z轴带轮驱动连接的转轴、固定套设在所述转轴上的驱动轴套、固接在所述Y轴滑块上的Z轴滑块、可滑动设置在所述Z轴滑块上且与所述加样机械臂固接的Z轴滑轨以及固接在所述加样机械臂上的用于与所述驱动轴套配合的驱动条，通过所述驱动轴套的转动带动所述驱动条沿Z轴方向运动。
可选地，所述荧光成像系统包括宽光谱光源、可切换荧光分光装置以及成像探测器，所述宽光谱光源多个LED光源，所述可切换荧光分光装置包括多个可切换进入光路的荧光分光模块；
所述宽光谱光源发出的激发光经所述荧光分光模块反射后到达样品，样品被激发产生的荧光透射所述荧光分光模块后进入所述成像探测器，实现荧光成像。
可选地，所述荧光成像系统还包括固接在所述安装座上的支撑架、固接在所述支撑架上的支撑板、设置在所述支撑板上的第二滑轨、可滑动设置在所述第二滑轨上的荧光切换滑板、第二主动带轮和第二从动带轮、设置在所述第二主动带轮和第二从动带轮之间的第二皮带以及设置在所述支撑板上且与所述第二主动带轮驱动连接的第二电机，所述荧光切换滑板与所述第二皮带连接。
可选地，所述支撑板上开设有成像孔，所述安装座可滑动至所述支撑板下方，以将所述安装座上的微流控芯片输送至所述成像孔下方，从而通过荧光成像系统对所述微流控芯片内的样品进行荧光成像。
可选地，所述支撑板上还设置有机架，所述成像探测器设置于所述机架上，且处于所述成像孔正上方；
所述多个荧光分光模块沿所述荧光切换滑板的滑动方向依次设置在所述荧光切换滑板上，通过所述荧光切换滑板一次将一个所述荧光分光模块运输至所述成像孔正上方，以切换进入光路；所述宽光谱光源设置在所述成像孔的侧部。
可选地，所述数据存储分析系统包括图像预处理与存储模块、荧光图像分割与定位模块以及数据统计分析模块；
所述荧光成像系统采集的荧光信号包括PCR扩增反应中每次热循环平台期的过程荧光图像以及PCR扩增反应完成后的最终荧光图像；
所述数据存储分析系统的处理方法包括以下步骤：
S1：所述图像预处理与存储模块对采集到的过程荧光图像和最终荧光图像进行预处理，并存储；
S2：所述荧光图像分割与定位模块根据所述微孔阵列芯片上的微孔位置建立图像网格模板，将所述步骤S1处理后的过程荧光图像、最终荧光图像分别与图像网格模板配准，实现过程荧光图像和最终荧光图像上微孔位置的定位；
S3：针对最终荧光图像，提取阳性信号样本所在的微孔位置信息，作为目标微孔位置；
S4：针对过程荧光图像，提取与所述目标微孔位置对应的阳性样本所在的微孔的荧光强度信息，并绘制阳性样本的实时荧光定量PCR曲线。
本申请的有益效果是：
本申请的超高通量的单细胞核酸实时荧光定量分析方法，可以实现十万量级、百万量级的单细胞捕获，通过多种荧光标记可实现多个基因位点的实时定量PCR分析检测，相比于现有产品，极大的提升了检测通量，并且实现了单个细胞的分析而非群体细胞分析；
本申请通过在微孔内修饰DNA探针捕获细胞内的目标核酸分子，可实现十万量级、百万量级的单细胞捕获、原位裂解、核酸扩增以及实时荧光定量曲线分析；
本申请先通过最终荧光图像来确定所有阳性样本的所在的微孔位置，然后通过过程荧光图像仅仅提取所有阳性样本每次热循环平台期的荧光图像，能减少非特异数据的计算，减小计算量，从而实现了超高通量实时荧光定量PCR曲线快速绘制。
本申请通过设计具有十万量级、百万量级微孔的芯片，并通过在微孔内修饰DNA探针捕获细胞内的目标核酸分子，可实现十万量级、百万量级的单细胞捕获，并进一步实现原位裂解、核酸扩增，能为超高通量单细胞核酸分子实时荧光定量分析提高芯片基础；
本申请通过对微孔阵列芯片进行亲、疏水处理，能增加微孔毛细力，利于单细胞吸入微孔，实现单细胞捕获；
本申请的微流控封装结构通过设计多歧路树状分叉结构的流道，能实现样品的均匀分布，并布满整个微孔阵列区，实现高效均匀进样，且能保证各个末端分支的流量基本一致，实现液面的平稳推进。
本申请的超高通量单细胞核酸分子实时荧光定量分析一体化快速检测系统，集成微流控芯片、自动加样装置、温控热循环装置、荧光成像系统以及数据存储分析系统，可实现样品的自动化检测处理，能十万量级、百万量级的单细胞捕获核酸扩增以及实时荧光定量曲线分析。
附图说明
图1为本申请的超高通量的单细胞核酸实时荧光定量分析方法的流程图；
图2为本申请的超高通量单细胞核酸分子实时荧光定量分析芯片的分解结构示意图；
图3为本申请的上层盖板的下表面的结构示意图；
图4为本申请的上层盖板的下表面设置有微孔阵列芯片时的结构示意图；
图5为本申请的底层基板上表面的副流道槽的结构示意图；
图6为本申请的微流控封装结构内的整体流道的结构示意图；
图7为本申请的超高通量单细胞核酸分子实时荧光定量分析一体化快速检测系统的结构示意图；
图8为本申请的自动加样装置的结构示意图；
图9为本申请的自动加样装置的另一个视角的结构示意图；
图10为本申请的Z轴驱动机构的局部结构示意图；
图11为本申请的温控热循环装置的结构示意图；
图12为本申请的温控热循环装置分解结构示意图；
图13为本申请的荧光成像系统的结构示意图；
图14为本申请的荧光成像系统的另一个视角的结构示意图；
图15为本申请的荧光分光模块的结构示意图；
图16为本申请的荧光成像系统的光路示意图。
附图标记说明：
5—微孔阵列芯片；6—微流控封装结构；50—微孔阵列区；60—底层基板；61—上层盖板；62—上芯片槽；63—进样流道槽；64—出样流道槽；65—副流道槽；66—下芯片槽；67—进样口；68—总出液口；69—缓冲液入口；70—缓冲液出口；71—树根节点端口；72—子节点端口；73—V型导流槽；
1—自动加样装置；
10—加样底座；11—X轴驱动机构；12—Y轴驱动机构；13—Z轴驱动机构；14—加样机械臂；
110—X轴滑轨；111—X轴主动带轮；112—X轴从动带轮；113—X轴皮带；114—X轴电机；115—加样安装板；
120—Y轴滑轨；121—Y轴主动带轮；122—Y轴从动带轮；123—Y轴皮带；124—Y轴电机；125—Y轴滑块；
130—Z轴电机；131—Z轴带轮；132—转轴；133—驱动轴套；134—Z轴滑块；135—Z轴滑轨；136—驱动条；137—转轴座；
140—加样针头；
2—温控热循环装置；20—安装座；21—温控底座；22—载物台；23—加热组件；24—导热器；25—散热组件；26—第一驱动机构；27—铜板；260—第一滑轨；261—第一滑块；262—第一主动带轮； 263—第一从动带轮；264—第一皮带；265—第一电机；
3—荧光成像系统；30—宽光谱光源；31—可切换荧光分光装置；32—成像探测器；33—支撑架；34—第二滑轨；35—荧光切换滑板；36—第二电机；37—第二主动带轮；38—第二从动带轮；39—第二皮带；300—第一LED光源；301—第二LED光源；302—第三LED光源；303—第一二向色镜；304—第二二向色镜；305—准直透镜；310—荧光分光模块；311—镜片安装块；312—激发滤光片；320—聚光镜；330—支撑板；331—成像孔；332—机架；
4—微流控芯片。
具体实施方式
下面结合实施例对本申请做进一步的详细说明，以令本领域技术人员参照说明书文字能够据以实施。
应当理解，本文所使用的诸如“具有”、“包含”以及“包括”术语并不排除一个或多个其它元件或其组合的存在或添加。
如图1所示，本实施例的一种超高通量的单细胞核酸实时荧光定量分析方法，包括以下步骤：
1)提供一种微孔阵列芯片，微孔阵列芯片上设置有至少一个微孔阵列区，微孔阵列区包括多个微孔，微孔内壁上修饰有至少一个DNA探针；
2)将待测样品加入微孔阵列芯片中，通过微孔捕获单细胞；
3)通过DNA探针捕获目标核酸分子；
4)进行PCR扩增检测，通过荧光定量分析，实现单细胞基因表达水平分析。
其中，微孔具有在一个微孔中只能容纳单个细胞的尺寸和形状。例如，在本实施例中，微孔的直径为1-100μm，形状为正六边形。且单个微孔阵列区内包括的微孔数量不小于10 6个，单个微孔阵列区能达到十万量级的单细胞捕获。进一步的，微孔阵列芯片上设置有10个一字排列的微孔阵列区，从而使微孔阵列芯片能达到总量为百万量级的超高通量单细胞捕获。当然，单个微孔阵列区的微孔数量以及微孔阵列芯片上的微孔阵列区的数量均可扩展，可实现千万量级的超高通量单细胞捕获。DNA探针用于捕获目标核酸分子，通过修饰多个DNA探针即可实现多个基因位点的检测。
其中，微孔阵列芯片的基底可采用聚合物、Si等材料，通过刻蚀，可形成直径在1μm-100μm的微孔结构。
本实施例中，微孔阵列芯片的表面进行了疏水修饰，对微孔内部进行亲水修饰，从而能增加微孔毛细力，减少表面液体残留。
其中，步骤3)中包括先向微孔阵列芯片中通入细胞裂解液，实现细胞原位裂解，通过微孔内修饰的DNA探针捕获目标核酸分子，然后再向微孔阵列芯片中通入缓冲液清洗。
其中，步骤4)中包括：向微孔阵列芯片中通入qPCR反应试剂，然后封闭微孔，进行PCR扩增，并通过荧光成像模块采集荧光信号，最后通过荧光定量分析模块进行荧光分析，以实现单细胞基因表达水平分析。微孔的封闭可采用油封，以实现各通道隔离；然后进入PCR反应装置中进行PCR扩增并通过荧光成像模块进行荧光检测。
微孔阵列芯片上设置有进样口、出样口、缓冲液入口和缓冲液出口。待测样品和qPCR反应试剂通过进样口进入，缓冲液入口和缓冲液出口供细胞裂解液和缓冲液等试剂的出入。
其中，荧光成像模块采集的荧光信号包括PCR扩增反应中每次热循环平台期的过程荧光图像以及PCR扩增反应完成后的最终荧光图像。即对于微孔阵列芯片的每一个区域，PCR扩增反应中，启动热循环后，在每一次热循环的低温退火的平台期均采集荧光图像，即为过程荧光图像。然后在整个PCR扩增反应完成后，再采集荧光图像即为最终荧光图像。通过最终荧光图像来确定所有阳性样本的所在的微孔位置，然后通过过程荧光图像提取所有阳性样本每次热循环平台期的荧光图像，以实现实时荧光定量PCR曲线绘制。
其中，荧光定量分析模块进行荧光分析的方法包括以下步骤：
S1：对过程荧光图像和最终荧光图像进行预处理，并存储；
S2：根据微孔阵列芯片上的微孔位置建立图像网格模板，调用步骤S1处理后的过程荧光图像、最终荧光图像分别与图像网格模板配准，实现过程荧光图像和最终荧光图像上微孔位置的分别定位，将过程荧光图像和最终荧光图像的位置对应起来并能获取到各个微孔的位置信息；
S3：针对最终荧光图像，提取所有阳性信号样本所在的微孔位置信息，作为目标微孔位置；
S4：针对过程荧光图像，提取与目标微孔位置对应的所有阳性样本所在的微孔的荧光强度信息，并绘制阳性样本的实时荧光定量PCR曲线。
其中，先通过最终荧光图像来确定所有阳性样本的所在的微孔位置，然后通过过程荧光图像仅仅 提取所有阳性样本每次热循环平台期的荧光图像，能减少非特异数据的计算，减小计算量，从而实现超高通量实时荧光定量PCR曲线快速绘制。
另外，进一步的实施例中，DNA探针用于捕获目标核酸分子，微孔中可通过修饰多种荧光标记的多个DNA探针即可实现多个基因位点的检测；与之对应的，荧光成像模块扩展为具有对多种荧光进行成像的功能，然后利用荧光定量分析模块对多种荧光信号进行分析，即可实现多个基因位点的检测。
如图2-6所示，本实施例的一种超高通量单细胞核酸分子实时荧光定量分析芯片，包括微孔阵列芯片5和微流控封装结构6，微孔阵列芯片5设置在微流控封装结构6内；
微孔阵列芯片5在其基底上设置有至少一个微孔阵列区50，微孔阵列区50具有多个微孔，微孔具有在一个微孔中只能容纳单个细胞的尺寸和形状，且微孔内壁上修饰有至少一个DNA探针。
其中，微孔阵列芯片5的基底可采用聚合物、Si等材料，通过刻蚀，可形成直径在1μm-100μm的微孔结构。且微孔为通孔，且为正多边形结构。单个微孔中只能容纳单个细胞，在本实施例中，微孔形状为正六边形，其外接圆的直径为1-100μm。且单个微孔阵列区50内包括的微孔数量不小于10 6个，单个微孔阵列区50能达到十万量级的单细胞捕获。进一步的，微孔阵列芯片5上设置有10个一字排列的微孔阵列区50，从而使微孔阵列芯片5能达到总量为百万量级的超高通量单细胞捕获。当然，单个微孔阵列区50的微孔数量以及微孔阵列芯片5上的微孔阵列区50的数量均可扩展，可扩展实现千万量级的超高通量单细胞捕获。其中的DNA探针用于捕获目标核酸分子，通过修饰多个DNA探针即可实现多个基因位点的检测。
其中，微孔的内表面经亲水处理，微孔阵列芯片5除微孔内表面的其他表面经疏水处理。通过微孔的内表面亲水处理，能增加微孔毛细力，利于单细胞吸入微孔；通过他表面疏水处理，能减少表面液体残留。处理后的微孔阵列芯片5能够通过微孔的毛细力吸入样本，实现单细胞捕获和原位观测。
其中，在一种优选的实施例中，微孔阵列芯片5的亲、疏水处理方法为：
1)亲水处理：将整个微孔阵列芯片5浸没在亲水试剂中反应一定时间，亲水试剂可采用双氧水、氨水、醋酸、浓硫酸、盐酸或氢氧化钠中的至少一种；
2)填充微孔：采用填充物填充微孔阵列芯片5的微孔，隔绝微孔内壁；填充物可采用水、三氯乙烯、正己烷、硅油、液体石蜡、固体石蜡、氟化油、蓝膜或PDMS中的至少一种；
3)疏水处理：将填充后的微孔阵列芯片5浸没在疏水试剂中反应一定时间，疏水化试剂可采用全氟癸基三甲氧基硅烷、十八烷基三氯硅烷、十八烷基三甲氧基硅烷、十六烷基三乙氧基硅烷、己基三乙氧基硅烷或辛基三氯硅烷中的至少一种；
4)去除微孔中的填充物：采用清洗试剂对微孔阵列芯片5进行超声清洗，将微孔中的填充物去除，然后烘干，得到处理完成的微孔阵列芯片5。清洗试剂可选用三氯乙烯、丙酮、乙醇或异丙醇。
本申请中主要为进行超高通量的单细胞核酸实时荧光定量分析提供一种超高通量的芯片，利用本申请的芯片进行荧光定量分析的主要步骤为：
1)制作本申请的芯片；
2)将待测样品加入微孔阵列芯片5中，通过微孔捕获单细胞，通过微孔中修饰的DNA探针捕获目标核酸分子；
3)进行PCR扩增检测，通过荧光定量分析，实现单细胞核酸实时荧光定量分析。
通过本申请的具有百万量级的微孔的芯片实现百万量级的超高通量单细胞捕获，从而为实现超高通量的单细胞核酸实时荧光定量分析方法和系统提供芯片支持。
本实施例中，微流控封装结构6包括底层基板60和上层盖板61，上层盖板61的下表面中部开设有上芯片槽62，上芯片槽62的两侧对称设置有与之连通的进样流道槽63和出样流道槽64；底层基板60上表面中部设置有下芯片槽66，下芯片槽66的一侧设置有与出样流道槽64的结构相同的副流道槽65；
上层盖板61与底层基板60贴合连接后，上芯片槽62和下芯片槽66位置正对形成用于容纳微孔阵列芯片5的芯片安装槽，进样流道槽63和底层基板60的上表面之间形成进样流道，出样流道槽64和副流道槽65位置正对形成出样流道。
本申请中利用微流控封装结构6封装微孔阵列芯片5，通过其流道的设计，可实现快速进样及样品的均匀分布。
其中，上层盖板61上设置有进样口67、总出液口68、缓冲液入口69和缓冲液出口70；进样流道槽63和出样流道槽64沿宽度方向设置在上层盖板61的下表面两侧，进样口67、总出液口68沿宽度方向设置在上层盖板61的两侧，缓冲液入口69和缓冲液出口70沿长度度方向设置在上层盖板61的两侧。
其中，进样流道槽63、出样流道槽64和副流道槽65结构相同，均为树状分叉结构流道，其具有 一个树根节点端口71和若干子节点端口72；
进样流道槽63的树根节点端口71与进样口67连通，其子节点端口72连通至上芯片槽62的靠近进样口67的一侧；
出样流道槽64的子节点端口72与上芯片槽62的靠近总出液口68的一侧连通，其树根节点端口71连通至总出液口68；
副流道槽65的子节点端口72与下芯片槽66的靠近总出液口68的一侧连通，其树根节点端口71连通至总出液口68；
下芯片槽66沿长度方向的两端分别与缓冲液入口69和缓冲液出口70连通。
微流控封装结构6内的整体流道的结构如图6所示。
样品在驱动泵的作用下，由进样口67进入，通过进样流道槽63均匀、快速分布到微孔阵列芯片5上表面，样品通过微孔的毛细力吸入微孔，从而实现微孔对单细胞的捕获，多余的样品从微孔阵列芯片5另一侧的出样流道槽64，经由总出液口68排出。
缓冲液经由缓冲液入口69进入下芯片槽66，到达微孔阵列芯片5下表面，从微孔底部与微孔内进行物质交换，多余的缓冲液可从缓冲液出口70排出。
进样时，由于毛细力的原因，微孔阵列芯片5上表面流入进微孔的样品需要克服毛细力作用才能从微孔底部流出流入下方区域，此时驱动泵的给样流速不足以使样品能克服微孔的毛细力作用，所以样品不会从微孔下部流出，保证细胞能固定在微孔内，同时也实现了微孔阵列芯片5上下区域的分层，可以实现上下两层的独立控制，可通入不同的试剂。当需要对微孔阵列芯片5的微孔进行冲洗清空时，总出液口68连接真空泵，清洗液从进样口67进入通过进样流道槽63分布到微孔阵列芯片5上表面，真空泵的抽吸作用使清洗液能克服微孔的毛细力作用，微孔中的液体可从微孔底部流出进入副流道槽65，然后由总出液口68排出。
本申请中采用树状分叉结构的流道，能实现样品的均匀分布，并布满整个微孔阵列区50，使每个微孔均能进样，且能保证各个末端分支的流量基本一致，实现液面的平稳推进。
更进一步的实施例中，树状分叉结构流道的子节点端口72的数量与微孔阵列芯片5上的微孔阵列区50的个数相同，均为10个。其中，树状分叉结构流道的子节点端口72设置有V型导流槽73，V型导流槽73的数量和位置与微孔阵列芯片5上的微孔阵列区50一一对应，数量均为10个，每一个V型导流槽73对应于一个微孔阵列区50的侧部；V型导流槽73的尖口端与子节点端口72连通，另一端与微孔阵列区50的侧部连通。通过V型导流槽73的设置，能利于液体的快速、均匀流动，以及分布和汇集。对于进样流道槽63而言，其子节点端口72的V型导流槽73利于将进入的样品快速、均匀的导入微孔阵列区50的上方。对于出样流道槽64和副流道槽65而言，其子节点端口72的V型导流槽73利于芯片安装槽中的液体快速汇集至子节点端口72中，以高效排出。
如图7所示，本实施例的一种超高通量单细胞核酸分子实时荧光定量分析一体化快速检测系统，包括：微流控芯片4、自动加样装置1、温控热循环装置2、荧光成像系统3以及数据存储分析系统；
自动加样装置1具有X轴、Y轴和Z轴方向的自由度，用于将样品和试剂自动加入微流控芯片4内；微流控芯片4设置在温控热循环装置2上；
温控热循环装置2用于实现微流控芯片4中的样品进行PCR扩增反应中的热循环温度控制；
荧光成像系统3用于采集样品的荧光信号并传输至数据存储分析系统；
数据存储分析系统对采集的样品的荧光信号进行分析，识别阳性样本，并绘制出阳性样本的实时荧光定量分析曲线。数据存储分析系统可内嵌于上位机(如计算机)中，上位机与荧光成像系统3通信连接，可接受荧光成像系统3采集的数据。
其中，微流控芯片4上设置有不小于10 6个微孔，微孔阵列设置，微孔具有在一个微孔中只能容纳单个细胞的尺寸和形状。
在一种优选的实施例中，微流控芯片4上设置有10个一字排列的微孔阵列区，每个微孔阵列区上设置有不小于10 6个微孔，整个微流控芯片4上设置有不少于10 7个微孔，从而使微孔阵列芯片能达到总量为百万量级的超高通量单细胞捕获。更为优选的实施例中，微孔内壁上修饰有至少一个DNA探针，DNA探针用于捕获目标核酸分子，通过修饰多个DNA探针即可实现多个基因位点的检测。更进一步的，微孔的直径为1-100μm，形状为正六边形。
其中，微孔阵列芯片的基底可采用聚合物、Si等材料，通过刻蚀，可形成直径在1μm-100μm的微孔结构。微流控芯片4上设置有常规的进样口、出样口、缓冲液入口和缓冲液出口。待测样品和qPCR反应试剂通过进样口进入，缓冲液入口和缓冲液出口供细胞裂解液和缓冲液等试剂的出入。
微流控芯片4中的样品在温控热循环装置2上实现PCR扩增，并通过荧光成像系统3进行荧光信 号采集。主要的工作过程为：将微流控芯片4放入温控热循环装置2，然后通过自动加样装置1向微流控芯片4中加入相关试剂，主要包括：先通入细胞裂解液，实现细胞原位裂解，通过微孔内修饰的DNA探针捕获目标核酸分子，然后通入缓冲液清洗；最后通入qPCR反应试剂，然后封闭微孔，进行PCR扩增，并通过荧光成像系统3采集荧光信号，最后通过数据存储分析系统进行荧光分析，以实现单细胞基因表达水平分析。其中，微孔的封闭可采用油封，以实现各通道隔离；然后进行PCR扩增和荧光检测。
自动加样装置1的加样方案、温控热循环装置2温控方案、荧光成像系统3的成像方案以及数据存储分析系统的分析处理方案可通过以下具体实施例实现。
其中，荧光成像模块系统的荧光信号包括PCR扩增反应中每次热循环平台期的过程荧光图像以及PCR扩增反应完成后的最终荧光图像。即对于微流控芯片4的每一个区域，PCR扩增反应中，启动热循环后，在每一次热循环的低温退火的平台期均采集荧光图像，即为过程荧光图像。然后在整个PCR扩增反应完成后，再采集荧光图像即为最终荧光图像。通过最终荧光图像来确定所有阳性样本的所在的微孔位置，然后通过过程荧光图像提取所有阳性样本每次热循环平台期的荧光图像，以实现实时荧光定量PCR曲线绘制。
在一种实施例中，数据存储分析系统的分析处理可通过以下方案实现：数据存储分析系统包括图像预处理与存储模块、荧光图像分割与定位模块以及数据统计分析模块；荧光成像系统3采集的荧光信号包括PCR扩增反应中每次热循环平台期的过程荧光图像以及PCR扩增反应完成后的最终荧光图像。
数据存储分析系统的处理方法包括以下步骤：
S1：图像预处理与存储模块对采集到的过程荧光图像和最终荧光图像进行预处理，并存储；
S2：荧光图像分割与定位模块根据微孔阵列芯片上的微孔位置建立图像网格模板，将步骤S1处理后的过程荧光图像、最终荧光图像分别与图像网格模板配准，实现过程荧光图像和最终荧光图像上微孔位置的定位；
S3：针对最终荧光图像，提取阳性信号样本所在的微孔位置信息，作为目标微孔位置；
S4：针对过程荧光图像，提取与目标微孔位置对应的阳性样本所在的微孔的荧光强度信息，并绘制阳性样本的实时荧光定量PCR曲线。
其中，先通过最终荧光图像来确定所有阳性样本的所在的微孔位置，然后通过过程荧光图像仅仅提取所有阳性样本每次热循环平台期的荧光图像，能减少非特异数据的计算，减小计算量，从而实现超高通量实时荧光定量PCR曲线的快速绘制。
实施例1
在上述实施例的基础上，参照图11和12，本实施例中，温控热循环装置2包括安装座20、可滑动设置在安装座20上的温控底座21、设置在温控底座21上的用于放置微流控芯片4的载物台22、设置在载物台22和温控底座21之间的加热组件23、设置在安装座20上的散热组件25以及用于驱动温控底座21在安装座20上滑动的第一驱动机构26。微流控芯片4放置在载物台22上，并通过透明盖板覆盖密封。
其中，更为优选的，温控底座21上设置有导热器24，导热器24具有多个导热鳍片，便于快速散热。导热器24上设置铜板27，加热装置为帕尔贴或热电偶，且设置在铜板27上，载物台22设置在加热装置上，通过加热装置对载物台22上的微流控芯片4中的样品进行加热。散热组件25为设置在安装座20上的多个散热风扇，通过散热风扇加速散热。
其中，第一驱动机构26包括设置在温控底座21上的第一滑轨260、设置在第一滑轨260上的第一滑块261、设置在安装座20上第一主动带轮262和第一从动带轮263、设置在第一主动带轮262和第一从动带轮263之间的第一皮带264以及与第一主动带轮262驱动连接的第一电机265；第一滑块261与第一皮带264连接，安装座20连接在第一滑块261上。通过第一电机265带动温控底座21沿X轴方向滑动，使温控底座21运输至散热风扇上方或是运离散热风扇上方。在开始加样和加试剂时，温控底座21处于初始位置，位于荧光成像系统3左侧。
进行PCR扩增反应和检测时，通过第一驱动机构26将温控底座21运输至反应位置，即荧光成像系统3下方，且处于散热风上方。需加热时，加热装置对样品加热；当需要降温时，下方的散热风扇启动，以快速降温。
实施例2
在上述实施例的基础上，参照图7-10，本实施例中，自动加样装置1包括固接在安装座20上的加 样底座10以及设置在加样底座10上的X轴驱动机构11、Y轴驱动机构12、Z轴驱动机构13、加样机械臂14；的X轴驱动机构11、Y轴驱动机构12、Z轴驱动机构13用于实现加样机械臂14沿X轴、Y轴和Z轴方向的运动。加样机械臂14上设置有加样针头140，可吸取样品、试剂等加入到微流控芯片4中。
更进一步的，X轴驱动机构11包括设置在加样底座10上的X轴滑轨110、X轴主动带轮111和X轴从动带轮112、设置在X轴主动带轮111和X轴从动带轮112之间的X轴皮带113、设置在加样底座10上且与X轴主动带轮111驱动连接的X轴电机114以及可滑动设置在X轴滑轨110上且与X轴皮带113连接的加样安装板115。X轴电机114电机通过X轴主动带轮111、X轴从动带轮112、X轴皮带113带动加样安装板115沿X轴方向运动，从而带动Y轴驱动机构12、Z轴驱动机构13、加样机械臂14整体沿X轴方向运动。
更进一步的，Y轴驱动机构12包括设置在加样安装板115上的Y轴滑轨120、Y轴主动带轮121和Y轴从动带轮122、设置在Y轴主动带轮121和Y轴从动带轮122之间的Y轴皮带123、设置在加样安装板115上且与Y轴主动带轮121驱动连接的Y轴电机124以及可滑动设置在Y轴滑轨120上且与Y轴皮带123连接的Y轴滑块125。Y轴电机124通过Y轴主动带轮121、Y轴从动带轮122、Y轴皮带123带动Y轴滑块125沿Y轴方向运动，从而带动加样机械臂14沿Y轴方向运动。
更进一步的，Z轴驱动机构13包括固接在加样安装板115上的Z轴电机130、与Z轴电机130驱动连接的Z轴带轮131、与Z轴带轮131驱动连接的转轴132、固定套设在转轴132上的驱动轴套133、固接在Y轴滑块125上的Z轴滑块134、可滑动设置在Z轴滑块134上且与加样机械臂14固接的Z轴滑轨135以及固接在加样机械臂14上的用于与驱动轴套133配合的驱动条136，通过驱动轴套133的转动带动驱动条136沿Z轴方向运动，Y轴滑块125上开设有供转轴132穿过的轴孔。Z轴带轮131包括与Z轴电机130驱动连接的主动轮、与主带轮通过皮带连接的从动轮，加样安装板115上设置有用于安装转轴132的转轴座137。Z轴电机130通过Z轴带轮131带动转轴132转动，转轴132上的驱动轴套133转动后带动驱动条136沿Z轴方向运动，从而实现加样机械臂14沿Z轴方向的运动。其中，驱动轴套133与驱动条136之间可采用摩擦驱动配合(如驱动轴套133为摩擦套或摩擦轮，驱动条136为摩擦板或或齿条板或摩擦面等，通过摩擦力，由驱动轴套133的转动带动驱动条136的Z向运动)，也可采用齿轮啮合(如驱动轴套133为齿轮套，外周设置齿部，驱动条136为齿条，通过齿轮套上的齿部与齿条啮合，将驱动轴套133的转动转换为驱动条136的Z向运动)。本实施例中，驱动轴套133为摩擦套，驱动条136为齿条板，通过摩擦套与齿条板之间的摩擦力，将摩擦套的转动转换为齿条板的Z向运动。
从而通过X轴驱动机构11、Y轴驱动机构12、Z轴驱动机构13能实现加样机械臂14沿X轴、Y轴和Z轴方向的运动，以实现对微流控芯片4的样品、各种试剂的自动添加。
实施例3
在上述实施例的基础上，参照图13-16，本实施例中，荧光成像系统3包括宽光谱光源30、可切换荧光分光装置31以及成像探测器32，宽光谱光源30多个LED光源，可切换荧光分光装置31包括多个可切换进入光路的荧光分光模块310；
宽光谱光源30发出的激发光经荧光分光模块310反射后到达样品，样品被激发产生的荧光透射荧光分光模块310后进入成像探测器32，实现荧光成像。当然宽光谱光源30还包括有分光光路，以将多个LED光源传输至荧光分光模块310。
作为优选的实施例，荧光成像系统3还包括固接在安装座20上的支撑架33、固接在支撑架33上的支撑板330、设置在支撑板330上的第二滑轨34、可滑动设置在第二滑轨34上的荧光切换滑板35、第二主动带轮37和第二从动带轮38、设置在第二主动带轮37和第二从动带轮38之间的第二皮带39以及设置在支撑板330上且与第二主动带轮37驱动连接的第二电机36，荧光切换滑板35与第二皮带39连接。
其中，支撑板330上开设有成像孔331，安装座20可滑动至支撑板330下方，以将安装座20上的微流控芯片4输送至成像孔331下方，从而通过荧光成像系统3对微流控芯片4内的样品进行荧光成像。
其中，支撑板330上还设置有机架332，成像探测器32设置于机架332上，且处于成像孔331正上方；
多个荧光分光模块310沿荧光切换滑板35的滑动方向依次设置在荧光切换滑板35上，通过荧光切换滑板35一次将一个荧光分光模块310运输至成像孔331正上方，以切换进入光路；宽光谱光源30设置在成像孔331的侧部。
在进一步的实施例中，宽光谱光源303个不同波长的LED光源，实现全波段可见光(波长为400nm-700nm)覆盖。分光光路包括设置在3个LED光源出射端的3个准直透镜305以及若干用于将LED光源发出的光反射或透射到荧光分光模块310上的二向色镜。例如，本实施例中，3个LED光源分别为第一LED光源300、第二LED光源301和第三LED光源302，二向色镜包括2个：第一二向色镜303和第二二向色镜304。第一LED光源300发出的光经过准直透镜305准直后依次透射第一二向色镜303和第二二向色镜304，进入荧光分光模块310；第二LED光源301发出的光经过准直透镜305准直后被第一二向色镜303反射，然后再透射第二二向色镜304，进入荧光分光模块310；第三LED光源302发出的光经过准直透镜305准直后被第二二向色镜304反射至荧光分光模块310。
其中，荧光分光模块310包括4个，每个荧光分光模块310均包括镜片安装块311以及设置在其中的激发滤光片312，每个激发滤光片312允许通过的波长各不相同，实现不同荧光的过滤。镜片安装块311四周均开孔。荧光分光模块310切换进光路时，荧光分光模块310处于成像孔331上方，且处于成像探测器32正下方，LED光源处于
成像探测器32选用高灵敏度CMOS相机或CCD相机，且相机前还设置有聚光镜320。宽光谱光源30发出的光经过分光光路后被荧光分光模块310反射至样品上，样品被激发产生的荧光再经过激发滤光片312后，经过聚光镜320到达CMOS相机，进行荧光成像。DNA探针用于捕获目标核酸分子，微孔中可通过修饰多种荧光标记的多个DNA探针即可实现多个基因位点的检测；与之对应的，荧光成像系统3需扩展为具有对多种荧光进行成像的功能，然后利用数据存储分析系统对多种荧光信号进行分析，即可实现多个基因位点的检测。所以本实施例中，通过设置三种不同波长的光源实现全波段可见光(波长为400nm-700nm)覆盖，并利用可切换的4个荧光分光模块310对不同的荧光进行过滤，最终对不同的荧光成像。
在一种实施例中，超高通量单细胞核酸分子实时荧光定量分析一体化快速检测系统的工作流程包括:
1)载物台22初始化，载物台22处于初始加样位置，将微流控芯片4装入载物台22，通过自动加样装置1向微流控芯片4中加入样品，通过微流控芯片4上的微孔结构产生的毛细力捕获单细胞；
2)利用自动加样装置1，通过缓冲液入口，加入细胞裂解液，实现细胞原位裂解，通过孔内修饰特异性DNA探针捕获目标核酸分子，然后通入缓冲液清洗；
3)通过自动加样装置1向微流控芯片4中加入qPCR反应试剂，通过油封实现各通道隔离，在微流控芯片4上覆盖透明盖板密封；
4)通过第一驱动机构26将载物台22整体移动至反应区(也即检测区)，微流控芯片4移动至成像孔331下方，启动PCR热循环，加热装置将样品加热至设定温度，并且在每个热循环平台期采集荧光图像，如使用了多种荧光标记则切换荧光分光模块310，逐一采集；然后通过载物台22移动，将微流控芯片4的下一个区域移动至成像孔331正下方，进行下一个区域的荧光成像；将采集到图像进行图像拼接形成一张包括微流控芯片4整个样品区域的荧光图像，即为该次热循环平台期的过程荧光图像。PCR扩增反应完成后，再采集微流控芯片4整个样品区域的荧光图像，即最终荧光图像；
5)数据存储分析系统调用采集的荧光信号，并进行分析，获得实时荧光定量PCR曲线，数据存储分析系统包括图像预处理与存储模块、荧光图像分割与定位模块以及数据统计分析模块，其具体分析方法包括：
5-1)：图像预处理与存储模块对采集到的过程荧光图像和最终荧光图像进行预处理(可包括图像拼接、降噪滤波、图像增强等)，并存储；
5-2)：荧光图像分割与定位模块根据微孔阵列芯片上的微孔位置建立图像网格模板，将步骤S1处理后的过程荧光图像、最终荧光图像分别与图像网格模板配准，实现过程荧光图像和最终荧光图像上微孔位置的定位；
5-3)：针对最终荧光图像，提取阳性信号样本所在的微孔位置信息，作为目标微孔位置；
5-4)：针对过程荧光图像，提取与目标微孔位置对应的阳性样本所在的微孔的荧光强度信息(即微孔位置的灰度均值)，并绘制阳性样本的实时荧光定量PCR曲线。其中，可应用多线程并行处理运算，同时提取多个微孔通道内的荧光强度，以提高处理速度。
上述过程为针对一种荧光的荧光信号处理，包括多种荧光信号时，按上述步骤分别处理即可。
尽管本申请的实施方案已公开如上，但其并不仅仅限于说明书和实施方式中所列运用，它完全可以被适用于各种适合本申请的领域，对于熟悉本领域的人员而言，可容易地实现另外的修改，因此在不背离权利要求及等同范围所限定的一般概念下，本申请并不限于特定的细节。

Claims (32)

  1. 一种超高通量的单细胞核酸实时荧光定量分析方法，其特征在于，包括以下步骤：
    1)提供一种微孔阵列芯片，所述微孔阵列芯片上设置有至少一个微孔阵列区，所述微孔阵列区包括多个微孔，所述微孔内壁上修饰有至少一个DNA探针；
    2)将待测样品加入所述微孔阵列芯片中，通过所述微孔捕获单细胞；
    3)通过所述DNA探针捕获目标核酸分子；
    4)进行PCR扩增检测，通过荧光定量分析，实现单细胞基因表达水平分析。
  2. 根据权利要求1所述的超高通量的单细胞核酸实时荧光定量分析方法，其特征在于，所述微孔具有在一个微孔中只能容纳单个细胞的尺寸和形状。
  3. 根据权利要求2所述的超高通量的单细胞核酸实时荧光定量分析方法，其特征在于，所述微孔的直径为1-100μm，形状为正六边形。
  4. 根据权利要求1所述的超高通量的单细胞核酸实时荧光定量分析方法，其特征在于，单个所述微孔阵列区内包括的微孔数量不小于10 6个。
  5. 根据权利要求4所述的超高通量的单细胞核酸实时荧光定量分析方法，其特征在于，所述微孔阵列芯片上设置有10个所述微孔阵列区。
  6. 根据权利要求1-5中任意一项所述的超高通量的单细胞核酸实时荧光定量分析方法，其特征在于，所述步骤1)中还包括对所述微孔阵列芯片的表面进行疏水修饰，对所述微孔内部进行亲水修饰。
  7. 根据权利要求6所述的超高通量的单细胞核酸实时荧光定量分析方法，其特征在于，所述步骤3)中包括先向所述微孔阵列芯片中通入细胞裂解液，实现细胞原位裂解，通过所述微孔内修饰的DNA探针捕获目标核酸分子，然后再向所述微孔阵列芯片中通入缓冲液清洗。
  8. 根据权利要求7所述的超高通量的单细胞核酸实时荧光定量分析方法，其特征在于，所述步骤4)中包括：向所述微孔阵列芯片中通入qPCR反应试剂，进行PCR扩增，并通过荧光成像模块采集荧光信号，最后通过荧光定量分析模块进行荧光分析，以实现单细胞基因表达水平分析。
  9. 根据权利要求8所述的超高通量的单细胞核酸实时荧光定量分析方法，其特征在于，所述荧光成像模块采集的荧光信号包括PCR扩增反应中每次热循环平台期的过程荧光图像以及PCR扩增反应完成后的最终荧光图像。
  10. 根据权利要求9所述的超高通量的单细胞核酸实时荧光定量分析方法，其特征在于，所述荧光定量分析模块进行荧光分析的方法包括以下步骤：
    S1：对过程荧光图像和最终荧光图像进行预处理，并存储；
    S2：根据所述微孔阵列芯片上的微孔位置建立图像网格模板，将所述步骤S1处理后的过程荧光图像、最终荧光图像分别与图像网格模板配准，实现过程荧光图像和最终荧光图像上微孔位置的定位；
    S3：针对最终荧光图像，提取阳性信号样本所在的微孔位置信息，作为目标微孔位置；
    S4：针对过程荧光图像，提取与所述目标微孔位置对应的阳性样本所在的微孔的荧光强度信息，并绘制阳性样本的实时荧光定量PCR曲线。
  11. 一种超高通量单细胞核酸分子实时荧光定量分析芯片，其特征在于，包括微孔阵列芯片和微流控封装结构，所述微孔阵列芯片设置在所述微流控封装结构内；
    所述微孔阵列芯片在其基底上设置有至少一个微孔阵列区，所述微孔阵列区具有多个微孔，所述微孔具有在一个微孔中只能容纳单个细胞的尺寸和形状，且所述微孔内壁上修饰有至少一个DNA探针。
  12. 根据权利要求11所述的超高通量单细胞核酸分子实时荧光定量分析芯片，其特征在于，所述微孔的内表面经亲水处理，所述微孔阵列芯片除所述微孔内表面的其他表面经疏水处理。
  13. 根据权利要求12所述的超高通量单细胞核酸分子实时荧光定量分析芯片，其特征在于，所述微孔阵列区包括一字排列在基底上的10个，单个所述微孔阵列区内包括的微孔数量不小于10 6个。
  14. 根据权利要求13所述的超高通量单细胞核酸分子实时荧光定量分析芯片，其特征在于，所述微 孔为通孔，且为正多边形结构。
  15. 根据权利要求14所述的超高通量单细胞核酸分子实时荧光定量分析芯片，其特征在于，所述微孔形状为正六边形，其外接圆的直径为1-100μm。
  16. 根据权利要求11-15中任意一项所述的超高通量单细胞核酸分子实时荧光定量分析芯片，其特征在于，所述微流控封装结构包括底层基板和上层盖板，所述上层盖板的下表面中部开设有上芯片槽，所述上芯片槽的两侧对称设置有与之连通的进样流道槽和出样流道槽；所述底层基板上表面中部设置有下芯片槽，所述下芯片槽的一侧设置有与所述出样流道槽的结构相同的副流道槽；
    所述上层盖板与底层基板贴合连接后，所述上芯片槽和下芯片槽位置正对形成用于容纳所述微孔阵列芯片的芯片安装槽，所述进样流道槽和所述底层基板的上表面之间形成进样流道，所述出样流道槽和副流道槽位置正对形成出样流道。
  17. 根据权利要求16所述的超高通量单细胞核酸分子实时荧光定量分析芯片，其特征在于，所述上层盖板上设置有进样口、总出液口、缓冲液入口和缓冲液出口；所述进样流道槽和出样流道槽沿宽度方向设置在所述上层盖板的下表面两侧，所述进样口、总出液口沿宽度方向设置在所述上层盖板的两侧，所述缓冲液入口和缓冲液出口沿长度度方向设置在所述上层盖板的两侧。
  18. 根据权利要求17所述的超高通量单细胞核酸分子实时荧光定量分析芯片，其特征在于，所述进样流道槽、出样流道槽和副流道槽结构相同，均为树状分叉结构流道，其具有一个树根节点端口和若干子节点端口；
    所述进样流道槽的树根节点端口与所述进样口连通，其子节点端口连通至所述上芯片槽的靠近所述进样口的一侧；
    所述出样流道槽的子节点端口与所述上芯片槽的靠近所述总出液口的一侧连通，其树根节点端口连通至所述总出液口；
    所述副流道槽的子节点端口与所述下芯片槽的靠近所述总出液口的一侧连通，其树根节点端口连通至所述总出液口；
    所述下芯片槽沿长度方向的两端分别与所述缓冲液入口和缓冲液出口连通。
  19. 根据权利要求18所述的超高通量单细胞核酸分子实时荧光定量分析芯片，其特征在于，所述树状分叉结构流道的子节点端口的数量与所述微孔阵列芯片上的微孔阵列区的个数相同。
  20. 根据权利要求19所述的超高通量单细胞核酸分子实时荧光定量分析芯片，其特征在于，所述树状分叉结构流道的子节点端口设置有V型导流槽，所述V型导流槽的数量和位置与所述微孔阵列芯片上的微孔阵列区一一对应；所述V型导流槽的尖口端与所述子节点端口连通，另一端与所述微孔阵列区的侧部连通。
  21. 一种超高通量单细胞核酸分子实时荧光定量分析一体化快速检测系统，其特征在于，包括：微流控芯片、自动加样装置、温控热循环装置、荧光成像系统以及数据存储分析系统；
    所述自动加样装置具有X轴、Y轴和Z轴方向的自由度，用于将样品和试剂自动加入所述微流控芯片内；所述微流控芯片设置在所述温控热循环装置上；
    所述温控热循环装置用于实现所述微流控芯片中的样品进行PCR扩增反应中的热循环温度控制；
    所述荧光成像系统用于采集样品的荧光信号并传输至所述数据存储分析系统；
    所述数据存储分析系统对采集的样品的荧光信号进行分析，识别阳性样本，并绘制出阳性样本的实时荧光定量分析曲线。
  22. 根据权利要求21所述的超高通量单细胞核酸分子实时荧光定量分析一体化快速检测系统，其特征在于，所述微流控芯片上设置有不小于10 6个微孔，所述微孔具有在一个微孔中只能容纳单个细胞的尺寸和形状。
  23. 根据权利要求22所述的超高通量单细胞核酸分子实时荧光定量分析一体化快速检测系统，其特征在于，所述微孔的直径为1-100μm，形状为正六边形。
  24. 根据权利要求21-23中任意一项所述的超高通量单细胞核酸分子实时荧光定量分析一体化快速检测系统，其特征在于，所述温控热循环装置包括安装座、可滑动设置在所述安装座上的温控底座、 设置在所述温控底座上的用于放置所述微流控芯片的载物台、设置在所述载物台和温控底座之间的加热组件、设置在所述安装座上的散热组件以及用于驱动所述温控底座在所述安装座上滑动的第一驱动机构。
  25. 根据权利要求24所述的超高通量单细胞核酸分子实时荧光定量分析一体化快速检测系统，其特征在于，所述第一驱动机构包括设置在所述温控底座上的第一滑轨、设置在所述第一滑轨上的第一滑块、设置在所述安装座上第一主动带轮和第一从动带轮、设置在所述第一主动带轮和第一从动带轮之间的第一皮带以及与所述第一主动带轮驱动连接的第一电机；所述第一滑块与所述第一皮带连接，所述安装座连接在所述第一滑块上。
  26. 根据权利要求25所述的超高通量单细胞核酸分子实时荧光定量分析一体化快速检测系统，其特征在于，所述自动加样装置包括固接在所述安装座上的加样底座以及设置在所述加样底座上的X轴驱动机构、Y轴驱动机构、Z轴驱动机构、加样机械臂；所述的X轴驱动机构、Y轴驱动机构、Z轴驱动机构用于实现所述加样机械臂沿X轴、Y轴和Z轴方向的运动。
  27. 根据权利要求26所述的超高通量单细胞核酸分子实时荧光定量分析一体化快速检测系统，其特征在于，所述X轴驱动机构包括设置在所述加样底座上的X轴滑轨、X轴主动带轮和X轴从动带轮、设置在所述X轴主动带轮和X轴从动带轮之间的X轴皮带、设置在所述加样底座上且与所述X轴主动带轮驱动连接的X轴电机以及可滑动设置在所述X轴滑轨上且与所述X轴皮带连接的加样安装板；
    所述Y轴驱动机构包括设置在所述加样安装板上的Y轴滑轨、Y轴主动带轮和Y轴从动带轮、设置在所述Y轴主动带轮和Y轴从动带轮之间的Y轴皮带、设置在所述加样安装板上且与所述Y轴主动带轮驱动连接的Y轴电机以及可滑动设置在所述Y轴滑轨上且与所述Y轴皮带连接的Y轴滑块；
    所述Z轴驱动机构包括固接在所述加样安装板上的Z轴电机、与所述Z轴电机驱动连接的Z轴带轮、与所述Z轴带轮驱动连接的转轴、固定套设在所述转轴上的驱动轴套、固接在所述Y轴滑块上的Z轴滑块、可滑动设置在所述Z轴滑块上且与所述加样机械臂固接的Z轴滑轨以及固接在所述加样机械臂上的用于与所述驱动轴套配合的驱动条，通过所述驱动轴套的转动带动所述驱动条沿Z轴方向运动。
  28. 根据权利要求27所述的超高通量单细胞核酸分子实时荧光定量分析一体化快速检测系统，其特征在于，所述荧光成像系统包括宽光谱光源、可切换荧光分光装置以及成像探测器，所述宽光谱光源多个LED光源，所述可切换荧光分光装置包括多个可切换进入光路的荧光分光模块；
    所述宽光谱光源发出的激发光经所述荧光分光模块反射后到达样品，样品被激发产生的荧光透射所述荧光分光模块后进入所述成像探测器，实现荧光成像。
  29. 根据权利要求28所述的超高通量单细胞核酸分子实时荧光定量分析一体化快速检测系统，其特征在于，所述荧光成像系统还包括固接在所述安装座上的支撑架、固接在所述支撑架上的支撑板、设置在所述支撑板上的第二滑轨、可滑动设置在所述第二滑轨上的荧光切换滑板、第二主动带轮和第二从动带轮、设置在所述第二主动带轮和第二从动带轮之间的第二皮带以及设置在所述支撑板上且与所述第二主动带轮驱动连接的第二电机，所述荧光切换滑板与所述第二皮带连接。
  30. 根据权利要求29所述的超高通量单细胞核酸分子实时荧光定量分析一体化快速检测系统，其特征在于，所述支撑板上开设有成像孔，所述安装座可滑动至所述支撑板下方，以将所述安装座上的微流控芯片输送至所述成像孔下方，从而通过荧光成像系统对所述微流控芯片内的样品进行荧光成像。
  31. 根据权利要求30所述的超高通量单细胞核酸分子实时荧光定量分析一体化快速检测系统，其特征在于，所述支撑板上还设置有机架，所述成像探测器设置于所述机架上，且处于所述成像孔正上方；
    所述多个荧光分光模块沿所述荧光切换滑板的滑动方向依次设置在所述荧光切换滑板上，通过所述荧光切换滑板一次将一个所述荧光分光模块运输至所述成像孔正上方，以切换进入光路；所述宽光谱光源设置在所述成像孔的侧部。
  32. 根据权利要求21所述的超高通量单细胞核酸分子实时荧光定量分析一体化快速检测系统，其特征在于，所述数据存储分析系统包括图像预处理与存储模块、荧光图像分割与定位模块以及数据统计分析模块；
    所述荧光成像系统采集的荧光信号包括PCR扩增反应中每次热循环平台期的过程荧光图像以及PCR扩增反应完成后的最终荧光图像；
    所述数据存储分析系统的处理方法包括以下步骤：
    S1：所述图像预处理与存储模块对采集到的过程荧光图像和最终荧光图像进行预处理，并存储；
    S2：所述荧光图像分割与定位模块根据所述微孔阵列芯片上的微孔位置建立图像网格模板，将所述步骤S1处理后的过程荧光图像、最终荧光图像分别与图像网格模板配准，实现过程荧光图像和最终荧光图像上微孔位置的定位；
    S3：针对最终荧光图像，提取阳性信号样本所在的微孔位置信息，作为目标微孔位置；
    S4：针对过程荧光图像，提取与所述目标微孔位置对应的阳性样本所在的微孔的荧光强度信息，并绘制阳性样本的实时荧光定量PCR曲线。
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