WO2020010786A1 - 一种细胞分析芯片及具有其的细胞荧光检测系统和检测方法 - Google Patents

一种细胞分析芯片及具有其的细胞荧光检测系统和检测方法 Download PDF

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WO2020010786A1
WO2020010786A1 PCT/CN2018/118707 CN2018118707W WO2020010786A1 WO 2020010786 A1 WO2020010786 A1 WO 2020010786A1 CN 2018118707 W CN2018118707 W CN 2018118707W WO 2020010786 A1 WO2020010786 A1 WO 2020010786A1
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cell
excitation light
excitation
flow channel
light source
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French (fr)
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邓宇
王文兵
郭钟宁
谭蓉
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Foshan Gewei Technology Co Ltd
Guangdong University of Technology
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Foshan Gewei Technology Co Ltd
Guangdong University of Technology
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6402—Atomic fluorescence; Laser induced fluorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells

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  • the invention relates to the technical field of cell analysis, and more particularly, to a cell analysis chip, a cell fluorescence detection system and a detection method having the same.
  • Lab-on-chip also referred to as microfluidic analysis chip, integrates basic operation units such as sample preparation, reaction, separation, and detection in biological and chemical medical analysis processes on a micron-scale chip to automatically complete analysis The whole process.
  • basic operation units such as sample preparation, reaction, separation, and detection in biological and chemical medical analysis processes
  • micron-scale chip to automatically complete analysis The whole process.
  • disciplines such as biology, chemistry, medicine, fluids, electronics, materials, and machinery.
  • microfluidic chips Since the introduction of microfluidic chips, their detection research has been a focus of attention, and various biological and chemical processes in microfluidic chips are usually completed at the two-micron level.
  • the detector is required to have high sensitivity, fast response speed, and miniature And other characteristics.
  • Fluorescence detection technology is a commonly used fluorescence detection technology on microfluidic chips. Because it does not require contact with the sample, non-destructive quantitative or qualitative detection is performed on the detection sample. Therefore, this technology is widely used to achieve high-sensitivity fluorescence.
  • the detection system needs to design a suitable fluorescence detection optical path. At present, orthogonal types, non-confocal types, confocal types, and parallel types are reported. Commonly used optical components include light sources, lenses, filters, dichroic mirrors, optical fibers, objective lenses, and photoelectric conversion elements.
  • the laser has the characteristics of high energy, good directivity, and easy focusing, it is particularly suitable as a detector excitation light source in a microfluidic chip to improve detection sensitivity.
  • the fluorescence detection systems in the prior art are usually only satisfied with the detection of samples with single-color fluorescence, and they cannot do anything with multi-color fluorescently labeled samples.
  • the existing multi-channel optical detection device can meet the needs of multi-color fluorescence detection, it often has certain shortcomings, resulting in low efficiency when it works.
  • the present invention provides a cell analysis chip, a cell fluorescence detection system and a detection method having the same.
  • the invention can greatly improve the detection efficiency, can meet the needs of multicolor fluorescence detection, the overall system design is simple, and the detection sensitivity and working efficiency of the entire system are improved.
  • a cell analysis chip which includes a first reaction cell, a second reaction cell, a flow channel and a buffer tank, the first reaction cell and the second reaction cell It communicates with one end of the flow channel through a duct, and the buffer pool is connected to the other end of the flow channel.
  • the flow channel has a serpentine reciprocating shape.
  • a serpentine reciprocating flow channel is provided between two adjacent reciprocating sections.
  • a plurality of well structures with two ends communicating with the adjacent two reciprocating sections respectively.
  • the end of the well structure near the first reaction cell is the entrance of the well structure
  • the end of the well structure near the buffer pool is the exit of the well structure
  • the diameter R of the well structure is 1.2- 1.5 times
  • the entrance opening width L1 of the well structure is 1.2-1.4 times the cell diameter
  • the exit opening width L2 of the well structure is 0.4-0.6 times the cell diameter.
  • the diameter L of the flow channel is 1.5-1.8 times the cell diameter, and the depth of the flow channel is 1.2-1.8 times the cell diameter.
  • a switch connecting the first reaction cell and the flow channel is provided with a switch.
  • the role of the cell analysis chip mainly includes the generation and capture of droplets.
  • the T-shaped cross-channel there have been many studies on the droplet generation process, and its hydrodynamic conditions are more clear.
  • the droplet generation process is as follows: Because the flow resistance of the liquid varies from place to place within the channel, the liquid flow will preferentially advance from the line with a small flow resistance.
  • the cell analysis chip of the present invention adopts a similar principle.
  • the first reaction cell is used for containing the cell suspension, which is a dispersed phase
  • the second reaction cell is used for containing the mineral oil, which is a continuous term.
  • the droplets are fixed in a well structure.
  • the invention also provides a cell fluorescence detection system, which includes a workstation, an excitation light source electrically connected to the workstation, a CCD, and a three-dimensional mobile platform.
  • the three-dimensional mobile platform is provided with a microfluidic chip.
  • the chip is the above-mentioned cell analysis chip.
  • An objective lens, a half mirror and a first light adjusting device are arranged in order from the bottom to the top of the microfluidic chip.
  • the CCD is provided above the first light adjusting device.
  • the position of the excitation light source is such that the light emitted by the excitation light source is reflected by the half mirror, and then focused by the objective lens on the microfluidic chip.
  • a first section is provided between the excitation light source and the half mirror. Two finishing devices.
  • the first light aligning device includes a cut-off filter and a focusing lens, and the focus lens is provided above the cut-off filter;
  • the second light aligning device includes a beam expander and an excitation filter, and the expander The beam mirror is disposed on a side close to the excitation light source, and the excitation filter is disposed on a side close to the half mirror.
  • the excitation light source is a multi-band excitation light source
  • the cut-off filter and the excitation filter are provided with cut-off filters and excitation filters whose numbers and types correspond to the wavelengths of the respective wavelength bands of the excitation light source, The switching of the cut-off filter and the excitation filter is controlled by the workstation.
  • the entire detection system is systemized and integrated according to a similar microscope structure, and the structure of each part of the optical path is stable, no complicated structural design is required, and the overall space size is smaller.
  • the invention also provides a method for detecting fluorescence of cells, which includes the following steps:
  • the microfluidic chip obtained in step S3 is fixed on a three-dimensional mobile platform.
  • the workstation controls the excitation light source to emit excitation light of a certain wavelength, and at the same time controls the cutoff filter and the excitation filter corresponding to the excitation light wavelength to enter the optical path to excite.
  • the excitation light from the light source is expanded by the beam expander, the stray light is filtered by the excitation filter, and finally the excitation light is reflected by the objective lens to focus on a certain area of the microfluidic chip through a half mirror.
  • the fluorescent dye corresponding to the wavelength of the excitation light carried by the cells on the area is excited to emit a fluorescent signal;
  • the objective lens collects the excited fluorescent signal, transmits the fluorescent signal after passing through the half mirror, filters out other stray light through the cut-off filter, and focuses the image to the CCD after focusing by the focusing lens, and generates a spectral signal to be detected.
  • the position and number information of the detected cells, and the position and number information of the detected cells are fed back to the workstation to complete the fluorescence detection of the cells in the area;
  • step S6 The workstation controls the movement of the three-dimensional mobile platform so that the excitation light is focused on another area of the same size on the microfluidic chip, and then step S5 is repeated to complete the fluorescence detection of the cell in the area;
  • step S7 Repeat step S6, so that all areas of the microfluidic chip can complete the fluorescence detection of the cells;
  • the workstation automatically switches the next excitation wavelength of the excitation light source, and simultaneously controls the corresponding excitation filter and cutoff filter to automatically switch into the optical path, and controls the microfluidic chip to return to the detection starting point for the detection of the next fluorescent signal;
  • the position and number information of all cells to be detected is collected by the workstation for subsequent information analysis.
  • the cell analysis chip of the present invention can separate and capture cells, and can change the chip structure design according to the size and size of the sample to be detected.
  • the excitation light source used in the detection system of the present invention has multiple excitation wavelengths, which can meet the needs of multicolor fluorescence detection.
  • the overall system design is relatively simple.
  • the detection system is systemized and integrated with a microscope-like structure. It has an autofocus function and an overall space. The size is small, the structure of each part of the optical path is stable, and the detection sensitivity and working efficiency of the entire system are improved.
  • the detection method of the invention adopts the area scanning detection form, which can adjust the size of the field of view according to different detection needs, which can greatly reduce the time required to perform the detection of a single well structure, and greatly improve the detection efficiency.
  • FIG. 1 is a schematic diagram of the overall structure of a cell analysis chip of the present invention.
  • FIG. 2 is a partially enlarged view at A in FIG. 1.
  • FIG. 3 is a schematic diagram of the working principle of the fluorescence detection system of the present invention.
  • FIG. 4 is a schematic structural diagram of a fluorescence detection system according to the present invention.
  • FIG. 5 is an actual cell distribution diagram after assuming detection in Embodiment 3 of the present invention.
  • FIG. 6 is an actual sample distribution diagram obtained by scanning the cells 1 with the excitation wavelength A in Embodiment 3 of the present invention.
  • FIG. 7 is an actual sample distribution diagram obtained by scanning the cells 2 using the excitation wavelength B in Embodiment 3 of the present invention.
  • a cell analysis chip includes a first reaction cell 1, a second reaction cell 2, a flow channel 3, and a buffer cell 4, and the first reaction cell 1 and the second reaction cell 2 communicates with one end of the flow channel 3 through a conduit, the buffer pool 4 is connected to the other end of the flow channel 3, the flow channel 3 is serpentine reciprocating, and the serpentine reciprocating flow channel 3 is adjacent to two A plurality of well structures 5 are provided between the reciprocating sections, the two ends of which are in communication with the adjacent two reciprocating sections, respectively.
  • the end of the well structure 5 near the first reaction cell 1 is the entrance of the well structure 5, and the end of the well structure 5 near the buffer pool 4 is the exit of the well structure 5.
  • the diameter R of the well structure 5 is 1.2-1.5 times the cell diameter.
  • the entrance opening width L1 of the well structure 5 is 1.2-1.4 times the cell diameter.
  • the exit opening width L2 of the well structure 5 is 0.4. -0.6 times.
  • the diameter L of the flow channel 3 is 1.5-1.8 times the cell diameter, and the depth of the flow channel 3 is 1.2-1.8 times the cell diameter.
  • a switch connecting the first reaction cell 1 and the flow channel 3 is provided with a switch.
  • the role of the cell analysis chip mainly includes the generation and capture of droplets.
  • the T-shaped cross-channel there have been many studies on the droplet generation process, and its hydrodynamic conditions are more clear.
  • the droplet generation process is as follows: Because the flow resistance of the liquid varies from place to place within the channel, the liquid flow will preferentially advance from the line with a small flow resistance.
  • the cell analysis chip of the present invention adopts a similar principle.
  • the first reaction cell 1 is used for containing a cell suspension, which is a dispersed phase
  • the second reaction cell 2 is used for containing a mineral oil, which is a continuous term.
  • the droplets are wrapped and fixed in the well structure 5.
  • a cell fluorescence detection system includes a workstation 6, an excitation light source 7, a CCD 8, and a three-dimensional mobile platform 9 electrically connected to the workstation 6.
  • the three-dimensional mobile platform 9 is provided with a microfluidic control.
  • Chip 10 the microfluidic chip 10 is the cell analysis chip described in Example 1, and the microfluidic chip 10 is provided with an objective lens 11, a semi-reflective lens 12 and a first light adjusting device in order from the bottom to the top.
  • the CCD 8 is disposed above the first light alignment device, and the position of the excitation light source 7 is such that the light emitted by the excitation light source 7 is reflected by the half mirror 12 and then focused by the objective lens 11 on the
  • the microfluidic chip 10 is provided with a second light adjusting device between the excitation light source 7 and the half mirror lens 12.
  • the first light adjusting device includes a cut-off filter 13 and a focusing lens 14, and the focusing lens 14 is disposed above the cut-off filter 13;
  • the second light adjusting device includes a beam expander 15 And an excitation filter 16, the beam expander 15 is provided on a side close to the excitation light source 7, and the excitation filter 16 is provided on a side close to the half mirror 12.
  • the excitation light source 7 is a multi-band excitation light source 7.
  • the cutoff filter 13 and the excitation filter 16 are provided with numbers and types corresponding to the wavelengths of the respective wavelengths of the excitation light source 7.
  • the cut-off filter and the excitation filter are controlled by the workstation 6.
  • the entire detection system is systemized and integrated according to a similar microscope structure.
  • the parts of the optical path are stable in structure, no complicated structural design is required, and the overall space size is smaller.
  • a method for detecting cell fluorescence including the following steps:
  • the microfluidic chip 10 obtained in step S3 is fixed on the three-dimensional mobile platform 9.
  • the workstation 6 controls the excitation light source 7 to emit excitation light of a certain wavelength, and simultaneously controls the cutoff filter and the excitation filter corresponding to the excitation light wavelength.
  • the excitation light from the excitation light source 7 is expanded by the beam expander 15 and then filtered by the excitation filter to remove stray light.
  • the reflection of the excitation light through the objective lens 11 is focused on the microfluidic through the half mirror 12 In a certain region of the chip 10, the fluorescent dye corresponding to the wavelength of the excitation light carried by the cells in the region is excited to emit a fluorescent signal;
  • the objective lens 11 collects the excited fluorescent signal, transmits the fluorescent signal after passing through the half mirror 12, and cuts off other stray light through the cut-off filter. After focusing by the focusing lens 14, it is imaged on the CCD 8 and generates a spectral signal. To obtain the position and number information of the detected cells, and feed back the position and number information of the detected cells to the workstation 6 to complete the fluorescence detection of the cells in the area;
  • step S6 The workstation 6 controls the movement of the three-dimensional mobile platform 9 so that the excitation light is focused on another area of the same size on the microfluidic chip 10, and then step S5 is repeated to complete the fluorescence detection of the cell in the area;
  • step S7 Repeat step S6, so that all areas of the microfluidic chip 10 can complete the fluorescence detection of the cells;
  • the workstation 6 automatically switches the next excitation wavelength of the excitation light source 7, and simultaneously controls the corresponding excitation filter and cut-off filter to automatically switch into the optical path, and controls the microfluidic chip 10 to return to the detection starting point to perform the next fluorescent signal.
  • the position and number information of all cells to be detected is collected by the workstation 6 for subsequent information analysis.
  • a five-color fluorescence detection system is taken as an example, and an area scanning detection form is adopted, and the visual field range is 4 * 4.
  • Sample capture is performed first, assuming that the actual cell distribution after detection is shown in Figure 5, 12345 represent five different cells. Different colors represent their fluorescence signals after being excited by light sources A, B, C, D, and E with different excitation wavelengths, respectively.
  • the cell 1 was scanned using the excitation wavelength A to locate a fluorescent signal color 1, and the actual sample distribution position can be obtained by the workstation 6 as shown in Figure 6. Through the calibration of the image, the fluorescence can be located Signal cell location.
  • the cell 2 is scanned using the excitation wavelength B to locate a fluorescent signal color 2, and the actual sample distribution position obtained by the workstation 6 is shown in FIG. 7.
  • the position of each type of fluorescent signal cell can be distinguished by scanning five times, and the position and number of different cells can be fed back for subsequent analysis.

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Abstract

一种细胞分析芯片（10）、具有细胞分析芯片（10）的荧光检测系统和检测方法，其中，细胞分析芯片（10）包括第一反应池（1）、第二反应池（2）、流道（3）和缓冲池（4），第一反应池（1）和第二反应池（2）通过导管与流道（3）的一端连通，缓冲池（4）与流道（3）另一端连接，流道（3）呈蛇形往复状，蛇形往复状的流道（3）相邻两往复段之间设有若干两端分别与该相邻两往复段连通的阱结构（5）。能够提升检测效率，能满足多色荧光检测的需求，系统整体设计简便，整个系统的检测灵敏度以及工作效率都得到提升。

Description

一种细胞分析芯片及具有其的细胞荧光检测系统和检测方法 技术领域
本发明涉及细胞分析技术领域，更具体地，涉及一种细胞分析芯片及具有其的细胞荧光检测系统和检测方法。
背景技术
随着现代生物医学技术的发展，以及人类对抗生素的滥用，传统的群体样本检测掩盖了大量重要信息，甚至会出现错误结果，需要对单个样本进行检测分析显得尤为重要，借助芯片实验室能很好完成样本检测分析。
芯片实验室-lab on a chip，又指微流控分析芯片，是把生物、化学医学分析过程的样品制备、反应、分离、检测等基本操作单元集成到一块微米尺度的芯片上，自动完成分析全过程。由于它在生物、化学、医学等领域的巨大潜力，已经发展成为一个生物、化学、医学、流体、电子、材料、机械等学科交叉的崭新研究领域。自微流控芯片问世以来，其检测研究是人们关注的热点，而微流控芯片中各种生物和化学过程都是通常在微米两级别完成，要求检测器具有灵敏度高、相应速度快、微型化等特点。
荧光检测技术是在微流控芯片上常用的荧光检测技术，由于其无需与样品接触，对检测样品进行非破坏性的定量或定性的检测，因此该技术被广泛使用，要实现高灵敏度的荧光检测系统，需要设计合适的荧光检测光路，目前报道的有正交型、非共聚焦型、共聚焦型、平行型等。常用的光学元件包括光源、透镜、滤光片、分色镜、光纤、物镜、光电转换元件等。
由于激光具有能量高、方向性好、易聚焦等特点，特别适合作为微流控芯片中检测器激发光源，以提高检测的灵敏度。然而现有技术中的荧光检测系统往往只满足于单色荧光对样品检测，对多色荧光标记的样本则无能为力。另外，现有的多通道光学检测装置虽然能满足多色荧光检测需求，但是其往往存在一定不足之处，导致其工作时效率低。
发明内容
本发明为克服上述现有技术所述的至少一种缺陷，提供一种细胞分析芯片及具有其的细胞荧光检测系统和检测方法。本发明能够大大提升检测效率，能满足 多色荧光检测的需求，系统整体设计简便，整个系统的检测灵敏度以及工作效率都得到提升。
为解决上述技术问题，本发明采用的技术方案是：一种细胞分析芯片，其中，包括第一反应池、第二反应池、流道和缓冲池，所述第一反应池和第二反应池通过导管与所述流道的一端连通，所述缓冲池与所述流道另一端连接，所述流道呈蛇形往复状，蛇形往复状的流道相邻两往复段之间设有若干两端分别与该相邻两往复段连通的阱结构。
进一步的，所述阱结构靠近所述第一反应池的一端为阱结构的入口，阱结构靠近所述缓冲池的一端为阱结构的出口，所述阱结构的直径R为细胞直径的1.2-1.5倍，所述阱结构的入口开口宽度L1为细胞直径的1.2-1.4倍，所述阱结构的出口开口宽度L2为细胞直径的0.4-0.6倍。
进一步的，所述流道的直径L为细胞直径的1.5-1.8倍，所述流道的深度为细胞直径1.2-1.8倍。
进一步的，连通所述第一反应池和流道的导管设有开关。
细胞分析芯片的作用主要包括液滴的生成及捕获两个单元，细胞分析芯片上通常应用的液滴的生成结构为两种，一种为T形交叉型通道，另一种为液流聚焦型通道，两种构形均利用连续相的剪切力将分散相剪断以生成液滴。对于T形交叉通道，液滴的生成过程的研究已经有很多，其流体动力学条件亦更加清楚。对于液流聚焦型通道，液滴的生成过程如下：由于液体的流阻在通道内各处不一，液流将优先从流阻小的线路前进，当前进受阻压力足够大时，受挤压通过通道狭窄处，当有稳定剂存在时，液滴能顺利通过而不分裂，最终稳定时大部分捕获单元将被液滴填充。本发明的细胞分析芯片采用类似的原理，第一反应池用于盛放细胞悬浮液，为分散相，第二反应池用于盛放矿物油，为连续项，利用液滴进行细胞的包裹并将液滴固定在阱结构中。
本发明还提供一种细胞荧光检测系统，其中，包括工作站、与所述工作站电连接的激发光源、CCD和三维移动平台，所述三维移动平台上设有微流控芯片，所述微流控芯片为上述的细胞分析芯片，所述微流控芯片上方由下往上依次设有物镜、半反半透镜和第一整光装置，所述CCD设在所述第一整光装置上方，所述激发光源的位置满足激发光源发出的光经所述半反半透镜反射后，再经所述物镜 聚焦在所述微流控芯片上，所述激发光源和半反半透镜之间设有第二整光装置。
进一步的，所述第一整光装置包括截止滤镜和聚焦透镜，所述聚焦透镜设在所述截止滤镜上方；所述第二整光装置包括扩束镜和激发滤镜，所述扩束镜设在靠近所述激发光源的一侧，所述激发滤镜设在靠近所述半反半透镜的一侧。
进一步的，所述激发光源为多波段的激发光源，所述截止滤镜和激发滤镜上设有数量和种类均与所述激发光源各波段的波长相对应的截止滤片和激发滤片，所述截止滤片和激发滤片的切换由所述工作站控制。
本发明中，整个检测系统按照类似显微镜结构系统化和集成化，光路各部分零件结构稳定，无需繁杂的结构设计，整体空间尺寸更小。
本发明还提供一种细胞荧光检测方法，其中，包括如下步骤：
S1.关闭连接第一反应池和流道的导管，向第二反应池中持续注入矿物油，待流道内部充满矿物油后，停止向第二反应池中注入矿物油，静置一定时间至流道与缓冲池连接的一端无液滴产生；
S2.将带有不同荧光染料标记的不同抗体与细胞悬浮液混合均匀，然后向第一反应池中注入细胞悬浮液，待第一反应池中充满细胞悬浮液后停止注入，然后缓缓打开连接第一反应池和流道的导管，静置一定时间以调整压力差；
S3.分别继续向第一反应池和第二反应池中注入细胞悬浮液和矿物油，控制第一反应池和第二反应池的流速，由于流道的宽度仅能容下一个细胞流过，因此细胞将一个接着一个流动并被阱结构所捕获，待所有阱结构中均捕获有细胞后，停止向第一反应池和第二反应池中注入细胞悬浮液和矿物油；
S4.将步骤S3得到的微流控芯片固定在三维移动平台上，工作站控制激发光源发出某一波长的激发光，同时控制与该激发光波长对应的截止滤片和激发滤片进入光路，激发光源发出的激发光经过扩束镜扩束后，再经过激发滤片滤去杂散光，最后通过半反半透镜将激发光反射经过物镜的作用聚焦在微流控芯片的某一区域上，该区域上的细胞所带的与激发光波长对应的荧光染料受激发发出荧光信号；
S5.物镜收集受激发的荧光信号，通过半反半透镜后将荧光信号透射出，通过截止滤片滤去其他杂散光，再经过聚焦透镜聚焦后成像到CCD上，并生成光谱信号，得到检测到的细胞的位置和数量信息，同时将检测到的细胞的位置和数量 信息反馈给工作站，完成该区域的细胞荧光检测；
S6.工作站控制三维移动平台移动，使得激发光聚焦在所述微流控芯片上另一个相同大小的区域上，然后重复步骤S5，完成该区域的细胞荧光检测；
S7.重复步骤S6，使得微流控芯片上所有区域均完成细胞荧光检测；
S8.工作站自动切换激发光源的下一个激发波长，同时控制相应的激发滤片和截止滤片自动切换进入光路，并控制微流控芯片回到检测起始点，进行下一种荧光信号的检测；依次类推，当与激发光源所有激发波长对应的受激发荧光信号检测完毕后，所有待检测细胞的位置和数量信息都被工作站收集，用于后续的信息分析。
与现有技术相比，本发明的有益效果：
本发明的细胞分析芯片能对细胞进行分离及捕获，可以根据待检测样品的大小和尺寸更改芯片结构设计。
本发明的检测系统所用的激发光源具有多个激发波长，能满足多色荧光检测的需求，系统整体设计较简便，采用类似显微镜结构将检测系统系统化和集成化，具有自动对焦功能，整体空间尺寸较小，光路各部分零件结构稳定，整个系统的检测灵敏度以及工作效率都得到提升。
本发明的检测方法采用区域扫描检测形式，可以根据不同检测需求来调整视野范围大小，能大大减少进行单个阱结构检测所需要的时间，检测效率大大提升。
附图说明
图1是本发明细胞分析芯片的整体结构示意图。
图2是图1中A处的局部放大图。
图3是本发明荧光检测系统的工作原理示意图。
图4是本发明荧光检测系统的结构示意图。
图5是本发明实施例3中假设检测完实际的细胞分布图。
图6是本发明实施例3中采用激发波长A对细胞①进行扫描得到的实际的样本分布图。
图7是本发明实施例3中采用激发波长B对细胞②进行扫描得到的实际的样本分布图。
具体实施方式
附图仅用于示例性说明，不能理解为对本专利的限制；为了更好说明本实施例，附图某些部件会有省略、放大或缩小，并不代表实际产品的尺寸；对于本领域技术人员来说，附图中某些公知结构及其说明可能省略是可以理解的。附图中描述位置关系仅用于示例性说明，不能理解为对本专利的限制。
实施例1
如图1和图2所示，一种细胞分析芯片，其中，包括第一反应池1、第二反应池2、流道3和缓冲池4，所述第一反应池1和第二反应池2通过导管与所述流道3的一端连通，所述缓冲池4与所述流道3另一端连接，所述流道3呈蛇形往复状，蛇形往复状的流道3相邻两往复段之间设有若干两端分别与该相邻两往复段连通的阱结构5。
如图1和图2所示，所述阱结构5靠近所述第一反应池1的一端为阱结构5的入口，阱结构5靠近所述缓冲池4的一端为阱结构5的出口，所述阱结构5的直径R为细胞直径的1.2-1.5倍，所述阱结构5的入口开口宽度L1为细胞直径的1.2-1.4倍，所述阱结构5的出口开口宽度L2为细胞直径的0.4-0.6倍。
如图1和图2所示，所述流道3的直径L为细胞直径的1.5-1.8倍，所述流道3的深度为细胞直径1.2-1.8倍。
本实施例中，连通所述第一反应池1和流道3的导管设有开关。
细胞分析芯片的作用主要包括液滴的生成及捕获两个单元，细胞分析芯片上通常应用的液滴的生成结构为两种，一种为T形交叉型通道，另一种为液流聚焦型通道，两种构形均利用连续相的剪切力将分散相剪断以生成液滴。对于T形交叉通道，液滴的生成过程的研究已经有很多，其流体动力学条件亦更加清楚。对于液流聚焦型通道，液滴的生成过程如下：由于液体的流阻在通道内各处不一，液流将优先从流阻小的线路前进，当前进受阻压力足够大时，受挤压通过通道狭窄处，当有稳定剂存在时，液滴能顺利通过而不分裂，最终稳定时大部分捕获单元将被液滴填充。本发明的细胞分析芯片采用类似的原理，第一反应池1用于盛放细胞悬浮液，为分散相，第二反应池2用于盛放矿物油，为连续项，利用液滴进行细胞的包裹并将液滴固定在阱结构5中。
实施例2
如图3所示，一种细胞荧光检测系统，其中，包括工作站6、与所述工作站 6电连接的激发光源7、CCD8和三维移动平台9，所述三维移动平台9上设有微流控芯片10，所述微流控芯片10为实施例1所述的细胞分析芯片，所述微流控芯片10上方由下往上依次设有物镜11、半反半透镜12和第一整光装置，所述CCD8设在所述第一整光装置上方，所述激发光源7的位置满足激发光源7发出的光经所述半反半透镜12反射后，再经所述物镜11聚焦在所述微流控芯片10上，所述激发光源7和半反半透镜12之间设有第二整光装置。
如图3所示，所述第一整光装置包括截止滤镜13和聚焦透镜14，所述聚焦透镜14设在所述截止滤镜13上方；所述第二整光装置包括扩束镜15和激发滤镜16，所述扩束镜15设在靠近所述激发光源7的一侧，所述激发滤镜16设在靠近所述半反半透镜12的一侧。
如图3所示，所述激发光源7为多波段的激发光源7，所述截止滤镜13和激发滤镜16上设有数量和种类均与所述激发光源7各波段的波长相对应的截止滤片和激发滤片，所述截止滤片和激发滤片的切换由所述工作站6控制。
本实施例中，整个检测系统按照类似显微镜结构系统化和集成化，如图4所示，光路各部分零件结构稳定，无需繁杂的结构设计，整体空间尺寸更小。
实施例3
一种细胞荧光检测方法，其中，包括如下步骤：
S1.关闭连接第一反应池1和流道3的导管，向第二反应池2中持续注入矿物油，待流道3内部充满矿物油后，停止向第二反应池2中注入矿物油，静置一定时间至流道3与缓冲池4连接的一端无液滴产生；
S2.将带有不同荧光染料标记的不同抗体与细胞悬浮液混合均匀，然后向第一反应池1中注入细胞悬浮液，待第一反应池1中充满细胞悬浮液后停止注入，然后缓缓打开连接第一反应池1和流道3的导管，静置一定时间以调整压力差；
S3.分别继续向第一反应池1和第二反应池2中注入细胞悬浮液和矿物油，控制第一反应池1和第二反应池2的流速，由于流道3的宽度仅能容下一个细胞流过，因此细胞将一个接着一个流动并被阱结构5所捕获，待所有阱结构5中均捕获有细胞后，停止向第一反应池1和第二反应池2中注入细胞悬浮液和矿物油；
S4.将步骤S3得到的微流控芯片10固定在三维移动平台9上，工作站6控制激发光源7发出某一波长的激发光，同时控制与该激发光波长对应的截止滤 片和激发滤片进入光路，激发光源7发出的激发光经过扩束镜15扩束后，再经过激发滤片滤去杂散光，最后通过半反半透镜12将激发光反射经过物镜11的作用聚焦在微流控芯片10的某一区域上，该区域上的细胞所带的与激发光波长对应的荧光染料受激发发出荧光信号；
S5.物镜11收集受激发的荧光信号，通过半反半透镜12后将荧光信号透射出，通过截止滤片滤去其他杂散光，再经过聚焦透镜14聚焦后成像到CCD8上，并生成光谱信号，得到检测到的细胞的位置和数量信息，同时将检测到的细胞的位置和数量信息反馈给工作站6，完成该区域的细胞荧光检测；
S6.工作站6控制三维移动平台9移动，使得激发光聚焦在所述微流控芯片10上另一个相同大小的区域上，然后重复步骤S5，完成该区域的细胞荧光检测；
S7.重复步骤S6，使得微流控芯片10上所有区域均完成细胞荧光检测；
S8.工作站6自动切换激发光源7的下一个激发波长，同时控制相应的激发滤片和截止滤片自动切换进入光路，并控制微流控芯片10回到检测起始点，进行下一种荧光信号的检测；依次类推，当与激发光源7所有激发波长对应的受激发荧光信号检测完毕后，所有待检测细胞的位置和数量信息都被工作站6收集，用于后续的信息分析。
本实施例中，以五色荧光检测系统为例，采用区域扫描检测形式，视野范围为4*4。先进行样品捕获，假设检测完实际的细胞分布如图5所示,①②③④⑤代表五种不同的细胞。不同颜色代表它们分别受不同激发波长光源A、B、C、D、E激发后的荧光信号。第一次扫描，采用激发波长A对细胞①进行扫描，定位出一种荧光信号颜色①，且工作站6能得到实际的样本分布位置如图6所示，通过图像的标定，可以定位出有荧光信号细胞的位置。第二次扫描，采用激发波长B对细胞②进行扫描，定位出一种荧光信号颜色②，且工作站6能得到实际的样本分布位置如图7所示。以此类推，扫描五次即可区分出每种荧光信号细胞的位置，并且不同细胞的位置和数量都能被反馈出来，用于后续的分析。
显然，本发明的上述实施例仅仅是为了清楚地说明本发明所作的举例，而并非是对本发明的实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。凡在本发明的精神和原则之内所作的任何修改、等同替换和改 进等，均应包含在本发明权利要求的保护范围之内。

Claims (8)

  1. 一种细胞分析芯片，其特征在于，包括第一反应池(1)、第二反应池(2)、流道(3)和缓冲池(4)，所述第一反应池(1)和第二反应池(2)通过导管与所述流道(3)的一端连通，所述缓冲池(4)与所述流道(3)另一端连接，所述流道(3)呈蛇形往复状，蛇形往复状的流道(3)相邻两往复段之间设有若干两端分别与该相邻两往复段连通的阱结构(5)。
  2. 根据权利要求1所述的一种细胞分析芯片，其特征在于，所述阱结构(5)靠近所述第一反应池(1)的一端为阱结构(5)的入口，阱结构(5)靠近所述缓冲池(4)的一端为阱结构(5)的出口，所述阱结构(5)的直径R为细胞直径的1.2-1.5倍，所述阱结构(5)的入口开口宽度L1为细胞直径的1.2-1.4倍，所述阱结构(5)的出口开口宽度L2为细胞直径的0.4-0.6倍。
  3. 根据权利要求1所述的一种细胞分析芯片，其特征在于，所述流道(3)的直径L为细胞直径的1.5-1.8倍，所述流道(3)的深度为细胞直径1.2-1.8倍。
  4. 根据权利要求1所述的一种细胞分析芯片，其特征在于，连通所述第一反应池(1)和流道(3)的导管设有开关。
  5. 一种细胞荧光检测系统，其特征在于，包括工作站(6)、与所述工作站(6)电连接的激发光源(7)、CCD(8)和三维移动平台(9)，所述三维移动平台(9)上设有微流控芯片(10)，所述微流控芯片(10)为权利要求1到3任一所述的细胞分析芯片，所述微流控芯片(10)上方由下往上依次设有物镜(11)、半反半透镜(12)和第一整光装置，所述CCD(8)设在所述第一整光装置上方，所述激发光源(7)的位置满足激发光源(7)发出的光经所述半反半透镜(12)反射后，再经所述物镜(11)聚焦在所述微流控芯片(10)上，所述激发光源(7)和半反半透镜(12)之间设有第二整光装置。
  6. 根据权利要求5所述的一种细胞荧光检测系统，其特征在于，所述第一整光装置包括截止滤镜(13)和聚焦透镜(14)，所述聚焦透镜(14)设在所述截止滤镜(13)上方；所述第二整光装置包括扩束镜(15)和激发滤镜(16)，所述扩束镜(15)设在靠近所述激发光源(7)的一侧，所述激发滤镜(16)设在靠近所述半反半透镜(12)的一侧。
  7. 根据权利要求6所述的一种细胞荧光检测系统，其特征在于，所述激发光源(7)为多波段的激发光源，所述截止滤镜(13)和激发滤镜(16)上设有数量和种类均与所述激发光源(7)各波段的波长相对应的截止滤片和激发滤片，所述截止滤片和激发滤片的切换由所述工作站(6)控制。
  8. 一种细胞荧光检测方法，其特征在于，包括如下步骤：
    S1.关闭连接第一反应池(1)和流道(3)的导管，向第二反应池(2)中持续注入矿物油，待流道(3)内部充满矿物油后，停止向第二反应池(2)中注入矿物油，静置一定时间至流道(3)与缓冲池(4)连接的一端无液滴产生；
    S2.将带有不同荧光染料标记的不同抗体与细胞悬浮液混合均匀，然后向第一反应池(1)中注入细胞悬浮液，待第一反应池(1)中充满细胞悬浮液后停止注入，然后缓缓打开连接第一反应池(1)和流道(3)的导管，静置一定时间以调整压力差；
    S3.分别继续向第一反应池(1)和第二反应池(2)中注入细胞悬浮液和矿物油，控制第一反应池(1)和第二反应池(2)的流速，待所有阱结构(5)中均捕获有细胞后，停止向第一反应池(1)和第二反应池(2)中注入细胞悬浮液和矿物油；
    S4.将步骤S3得到的微流控芯片(10)固定在三维移动平台(9)上，工作站(6)控制激发光源(7)发出某一波长的激发光，同时控制与该激发光波长对应的截止滤片和激发滤片进入光路，激发光源(7)发出的激发光经过扩束镜(15)扩束后，再经过激发滤片滤去杂散光，最后通过半反半透镜(12)将激发光反射经过物镜(11)的作用聚焦在微流控芯片(10)的某一区域上，该区域上的细胞所带的与激发光波长对应的荧光染料受激发发出荧光信号；
    S5.物镜(11)收集受激发的荧光信号，通过半反半透镜(12)后将荧光信号透射出，通过截止滤片滤去其他杂散光，再经过聚焦透镜(14)聚焦后成像到CCD上，并生成光谱信号，得到检测到的细胞的位置和数量信息，同时将检测到的细胞的位置和数量信息反馈给工作站(6)，完成该区域的细胞荧光检测；
    S6.工作站(6)控制三维移动平台(9)移动，使得激发光聚焦在所述微流控芯片(10)上另一个相同大小的区域上，然后重复步骤S5，完成该区域的细胞荧光检测；
    S7.重复步骤S6，使得微流控芯片(10)上所有区域均完成细胞荧光检测；
    S8.工作站(6)自动切换激发光源(7)的下一个激发波长，同时控制相应的激发滤片和截止滤片自动切换进入光路，并控制微流控芯片(10)回到检测起始点，进行下一种荧光信号的检测；依次类推，当与激发光源(7)所有激发波长对应的受激发荧光信号检测完毕后，所有待检测细胞的位置和数量信息都被工作站(6)收集，用于后续的信息分析。
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