CN100497653C - Method for analyzing single cell inclusion based on micro flow-controlled chip - Google Patents

Method for analyzing single cell inclusion based on micro flow-controlled chip Download PDF

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CN100497653C
CN100497653C CN 02144772 CN02144772A CN100497653C CN 100497653 C CN100497653 C CN 100497653C CN 02144772 CN02144772 CN 02144772 CN 02144772 A CN02144772 A CN 02144772A CN 100497653 C CN100497653 C CN 100497653C
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cell
cells
waste reservoir
single cell
inclusion
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CN1508261A (en )
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刘晓君
戴忠鹏
林炳承
盖宏伟
马银法
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中国科学院大连化学物理研究所
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一种基于微流控芯片的单细胞内涵物的分析方法,其特征在于;整个过程在一芯片平台上进行:首先将细胞池和细胞废液池分别加电压和接地,在电动力的驱动下,细胞由细胞池流向细胞废液池,在显微镜观察下,当单个细胞到达检测通道的入口时,将细胞池和细胞废液池电极悬浮;与此同时将运行缓冲液池、细胞裂解液池加高电压,废液池接地;在该种电压模式下,保障细胞、细胞裂解液、筛分剂、运行缓冲液并行进入检测通道,完成单细胞的裂解,分离;在检测通道的两端对内涵物进行检测。 A method of analysis of inclusion microfluidic chip based on a single cell, which is characterized in that; the whole process on a chip platform: first pool of cells and cell waste reservoir were applied and the ground voltage, the driving force in , flows from the pools of cells cells cells waste reservoir, under microscopic observation, when a single cell to the inlet of the detection channel, the pools of cells and cell waste reservoir electrode suspension; while the running buffer pools, pool of cell lysate high voltage, a waste reservoir is grounded; in that mode voltage, to protect cells, cell lysates, screening agents, parallel running buffer into the detection channel, complete lysis of single cells separated; at both ends of the detection channel connotation was detected. 本发明既可以单独分析单个细胞内的核酸、蛋白质、氨基酸、糖及其它物质,也可以并行分析上述物质,同时该方法具有相当的通量,克服了毛细管电泳技术耗时长的问题。 The present invention may be either a separate analysis of nucleic acid, protein, amino acids, sugars and other substances within individual cells, these substances may be analyzed in parallel, while the process has considerable flux, the capillary electrophoresis technique overcomes the problem of time consuming.

Description

一种基于微流控芯片的单细胞内涵物的分析方法 Analysis of inclusion single cell based microfluidic chips

技术领域: FIELD:

本发明涉及单细胞内涵物的分析方法,特别提供了一种基于微流控芯片的分析方法。 The present invention relates to a method for analysis of inclusion of a single cell, in particular to provide a method of analysis based on microfluidic chips. 背景技术: Background technique:

传统的分析细胞内涵物方法以大量细胞为前提,获取某一内涵物(比如蛋白质,核酸等)含量的总值,该值与细胞数量的比值外推至单细胞内该物质的含量。 Traditional methods of inclusion in the analysis cell large cell premise of inclusion obtain a (such as proteins, nucleic acids, etc.) the total value of the content, and the outside of the cell number ratio of values ​​pushed to the content of the substance in a single cell. 对于性质相对均一的细胞群来说,此方法可以接受,但在另外一些场合下,比如某些重大疾病的早期阶段,仅有个别细胞的组分发生变化,这时,传统方法将会平均掉该特异变化。 The nature of a relatively uniform population of cells, the method can be acceptable, but in other occasions, such as early stages of some of the major diseases, only individual components of the cell is changed, this time, the conventional method will average out the specific changes. 因此,单个细胞层面的分析有助于疾病的早期诊断。 Therefore, the analysis of a single cell level help in the early diagnosis of the disease. 此外,与传统方法相比,单细胞分析在信息的获取上相对准确;有可能检测活性周期较短的不稳定中间产物。 Further, as compared with the conventional method, the analysis of single cell obtaining relatively accurate information; it is possible to detect a short period of activity unstable intermediate.

从技术角度来看,单个细胞的分析可以分为完整的单细胞分析和破碎的单细胞分析。 From a technical standpoint, the individual cells can be divided into complete single cell analysis and fragmentation of single cell analysis. 本发明仅涉及到后一种技术。 The present invention relates only to the latter technique. 在该技术类别中目前已有的技术包括单细胞凝胶电泳和毛细管电泳。 In the technology category currently available technologies, including single-cell gel electrophoresis and capillary electrophoresis. 前者的对象更多的局限于DNA损伤的分析。 The former object more limited analysis of DNA damage. 毛细管电泳技术分析单细胞内涵物的方法相对比较成熟,但它存在着操作困难,通量低,不易完全自动化等缺点。 The method of capillary electrophoresis analysis of a single cell of inclusion of relatively mature, but there are difficulties in operation, low throughput, easy to fully automate and other shortcomings.

微流控芯片作为一种新型分析平台(又名芯片实验室,微全分析系统等)具有易自动化,集成化程度高等优势,其在单细胞内涵物分析检测方面的工作,世界范围内尚未展开。 Microfluidic chip as a new analysis platform (also known as chip laboratory, micro total analysis systems, etc.) are easy automation, higher degree of integration advantages of its work at the single cell analysis and detection aspects of inclusion, the world has not yet commenced . 仅有文献报道[Anal.Chem.1997, 69,1564-1568, Paul CH Li and D. Jed. Harrison]细胞在玻璃通道内的TBE缓冲液中迁移和在SDS中的裂解,但没有完成分离和检测的工作。 Only literature [Anal.Chem.1997, 69,1564-1568, Paul CH Li and D. Jed. Harrison] TBE buffer cell within the glass channel migration and lysed in the SDS, but did not complete the separation and work detection.

发明内容: SUMMARY:

本发明的目的在于提供一种以微流控芯片为操作平台的单细胞内涵物的分析方法,该方法既可以单独分析单个细胞内的核酸、蛋白质、氨基酸、 糖及其它物质,也可以并行分析上述物质,同时该方法具有相当的通量, 克服了毛细管电泳技术耗时长的问题。 Object of the present invention is to provide a method of analysis in microfluidic chip platform of inclusion of a single cell, the method may be analyze nucleic acids, proteins, amino acids, sugars and other substances within individual cells separately, may be analyzed in parallel thereof, while the process has considerable flux, the capillary electrophoresis technique overcomes the problem of time consuming.

本发明提供了一种基于微流控芯片的单细胞内涵物的分析方法,其特征在于;整个过程在一芯片平台上进行: The present invention provides a method based on single cell analysis of inclusion microfluidic chip, characterized in that; the whole process on a chip platform:

芯片平台上设置有细胞池、细胞废液池、细胞裂解液池、运行缓冲液池、废液池和检测通道,细胞池、细胞废液池、细胞裂解液池、缓冲液池分别与检测通道的入口相通,检测通道的出口接废液池,通道中预先靠压力充满筛分剂和染料; It is provided with a cell pools, waste reservoir cell, reservoir cell lysate, running buffer pools, waste reservoir and detection channels, cell pools, waste reservoir cell, reservoir cell lysate, a buffer pool for the detection on the channel chip platform an inlet in communication, connected to the outlet of the detection channel waste reservoir, the channel is filled in advance by the pressure screening agents and dyes;

首先将细胞池和细胞废液池分别加电压和接地,在电动力的驱动下, 细胞由细胞池流向细胞废液池,在显微镜观察下,当单个细胞到达检测通道的入口时,将细胞池和细胞废液池电极悬浮; Cell pools and cell first waste reservoir and the ground voltage are applied, in the driving force, flows from pools of cells cells cells waste reservoir, under microscopic observation, when a single cell to the inlet of the detection channel, the cell pool electrode suspension and cell waste reservoir;

与此同时将运行缓冲液池、细胞裂解液池加高电压,废液池接地;在该种电压模式下,保障细胞、细胞裂解液、筛分剂、运行缓冲液并行进入检测通道,完成单细胞的裂解及内涵物的分离; At the same time the running buffer pools, pool high voltage cell lysate, a waste reservoir is grounded; In that mode voltage, to protect cells, cell lysates, screening agents, parallel running buffer into the detection channel, to complete a single separating the cleaved and cells of inclusion;

在检测通道的两端对内涵物进行检测; Connotation was detected at both ends of the detection channel;

其中所述细胞浓度应在10S个/mL-l(^个/mL之间;筛分剂浓度应在0.15%-10%之间。 本发明中细胞最好选用各种没有细胞壁的细胞。细胞裂解液可以选用任何已知的裂解液,如十二烷基磺酸钠、十二垸基硫酸钠、十四烷基硫酸钠、十二烷基三甲基氯化铵、十二甲基三甲基溴化铵、聚氧化乙烯-23-十二垸基醚(Brp5)、辛基葡萄糖苷、Triton X-100等),筛分剂包括羟丙基甲基纤维素(HPMC),羟乙基纤维素(HEC),聚丙烯酰胺(PA),聚吡咯烷酮(PVP),聚环氧乙烷(PEO),聚乙烯醇(PVA),甲基纤维素(MC), 聚糖等。 Wherein the cell concentration should be in a 10S / mL-l (^ cells / mL between; screening agent concentration should be between 0.15% and 10% of cells in the present invention preferably use a variety of cells without cell walls. Lysates can use any known lysate, such as sodium dodecyl sulfate, dodecyl alkyl with sodium tetradecyl sulfate, dodecyl trimethyl ammonium chloride, dodecyl methyl tris bromide, polyoxyethylene -23- twelve embankment ether (Brp5), octyl glucoside, Triton X-100, etc.), screening agents include hydroxypropyl methyl cellulose (HPMC), hydroxyethyl acetate cellulose (HEC), polyacrylamide (PA), polyvinylpyrrolidone (PVP), polyethylene oxide (PEO), polyvinyl alcohol (PVA), methylcellulose (MC), polysaccharides and the like.

本发明中所用细胞浓度应在1(^个/mL-l(^个/mL之间,细胞浓度低于这个范围,耗时长。高于这个范围,由于细胞相互接触的机会大,难以做到单细胞分析。单细胞细胞裂解液浓度应在0.01%-0.5%之间,裂解液浓度低于该范围,细胞在流过通道时仍保持完整。高于此范围,细胞裂解过'决。 筛分剂浓度应在0.15%-10%之间,低浓度的筛分介质达不到筛分效果,高浓度的筛分介质粘度大,难以灌入通道中。 The cell concentration in the present invention should be between (a ^ / mL-l (^ / mL, and cells in a concentration below this range, time-consuming. Above this range with, due to the large cell chance of mutual contact, difficult to achieve remain single cell analysis. single cell lysis solution concentration should be between 0.01% to 0.5%, the concentration of lysate is below this range, the complete cell through the passage. above this range, cell lysis through 'decision sieve sub-agent concentration should be between 0.15% and 10%, lower concentration than the screening effect of the screening medium, large sieving medium viscosity and high concentration, it is difficult poured into the channel.

本发明可以用于单细胞内核酸,蛋白质,氨基酸,糖等物质的分离分析。 The present invention may be used in a single cell separation and analysis of nucleic acid species, proteins, amino acids, sugars and the like.

本发明用微流控芯片材料可以是石英、玻璃、硅或者PMMA、 PDMS 聚合物。 The present invention is a microfluidic chip material may be quartz, glass, silicon, or PMMA, PDMS polymers. 具体实施方式: detailed description:

图1为一种单细胞内涵物分析用微流控芯片,当然该结构并不限制本发明,其中1为细胞池、2为细胞废液池、3为细胞裂解液池、4为运行缓冲液池、5为废液池、6为检测点I 、 7为检测点II 。 Figure 1 is a single-cell analysis of inclusion microfluidic chip, of course, this structure does not limit the present invention, wherein the cell is a cell, cell 2 is a waste reservoir, a cell lysate pool 3, 4 running buffer pools, waste reservoir 5, 6 to detect a point I, 7 for the detection point II. 以激光诱导荧光的方法进行检测:首先向各缓冲池中分别加入缓冲液, 细胞裂解液,筛分剂(其中包含染料),细胞液。 In the method of laser-induced fluorescence detection: First, each buffer pool buffer were added, cell lysate, screening agents (which contains the dye), the cell fluid. 随后,在各缓冲池中插入 Subsequently, inserted in the buffer pool

铂电极,电高压(该电压在通道内产生的场强在50V/cm-250V/cm之间)通过该电极施加到各缓冲池,驱动细胞、细胞裂解液、筛分剂流向同一个检测通道。 Platinum electrode, electric high-voltage (the voltage generated in the passage between the field strength 50V / cm-250V / cm) is applied through the electrode to the respective pools, the drive cells, cell lysates, with a screening agent flow detection channel . 在该检测通道内完成单个细胞的裂解及释放的内涵物与染料的标记。 Completion of a single cell in the detection channel cleavage and release of the dye labeled connotation. 当标记了染料的内涵物通过激光焦斑时,被激光激发出的荧光由光电倍增管采集。 When the dyes of inclusion marked by the laser focal spot, the laser excited fluorescence collected by the photomultiplier tube. 实施例1 Example 1

见附图1芯片结构图。 See Figure 1 chip architecture of FIG. 运行缓冲液为100mM TBE,细胞为PC12 (大鼠肾上腺嗜咯细胞瘤),浓度约105个/mL。 The running buffer was 100mM TBE, in PC12 cells (rat adrenal slightly cell tumor), a concentration of about 105 / mL. 细胞裂解液为1%SDS。 Cell lysate of 1% SDS. 所用染料是YOYO-I (0.3uM)。 The dye is YOYO-I (0.3uM). 激光波长488nm。 Laser wavelength 488nm. 细胞在裂解池和废液池之间的通道内裂解,释放出的RNA带负电,所以在检测点I处检测。 Cells were lysed in a passage between the reservoir and the waste reservoir cleaved, releasing RNA negatively charged, the detection at the detection point I. 当检测带正电的物质时,可以在检测点II处检测。 When detecting positively charged substances, may be detected at the detection point II. 检测的RNA如附图2所示。 RNA was detected as shown in FIG. 2.

实施例2 Example 2

见附图1所示芯片结构图。 Chip structure shown in FIG. 1 see Fig. 运行缓冲液为100mM TBE,细胞为PC12 (大鼠肾上腺嗜咯细胞瘤),浓度约10S个/mL。 The running buffer was 100mM TBE, in PC12 cells (rat adrenal slightly cell tumor), a concentration of about 10S / mL. 细胞裂解液为1%SDS。 Cell lysate of 1% SDS. 筛分剂为1。 Screening agent is 1. /。 /. HPMC (50cp)。 HPMC (50cp). 激光波长488nm。 Laser wavelength 488nm. 在细胞液池,运行缓冲液池, 细胞裂解液池施加500V电压,废液池接地。 In the cell liquid pool, running buffer pools, pool of cell lysate was applied to a 500V, waste reservoir is grounded. 在检测点I处检测。 I detected at the detection point. 检测的RNA如附图3所示。 RNA was detected as shown in FIG. 3.

Claims (6)

  1. 1、一种基于微流控芯片的单细胞内涵物的分析方法,其特征在于;整个过程在一芯片平台上进行:芯片平台上设置有细胞池、细胞废液池、细胞裂解液池、运行缓冲液池、废液池和检测通道,细胞池、细胞废液池、细胞裂解液池、缓冲液池分别与检测通道的入口相通,检测通道的出口接废液池,通道中预先靠压力充满筛分剂和染料;首先将细胞池和细胞废液池分别加电压和接地,在电动力的驱动下,细胞由细胞池流向细胞废液池,在显微镜观察下,当单个细胞到达检测通道的入口时,将细胞池和细胞废液池电极悬浮;与此同时将运行缓冲液池、细胞裂解液池加高电压,废液池接地;在该种电压模式下,保障细胞、细胞裂解液、筛分剂、运行缓冲液并行进入检测通道,完成单细胞的裂解及内涵物的分离;在检测通道的两端对内涵物进行检测;其中所 1, a method of analysis of inclusion microfluidic chip based on a single cell, which is characterized in that; the whole process on a chip platform: the platform is provided with on-chip cell pools, waste reservoir cell, reservoir cell lysate, running buffer reservoir, a waste reservoir and detection channels, cell pools, waste reservoir cell, reservoir cell lysate, a buffer pool for detection channel communicating with the inlet, an outlet connected to a waste reservoir of the detection channel, the channel is filled in advance by a pressure screening agents and dyes; first pool of cells and cell waste reservoir were applied voltage and ground, driven by electric power, the flow of cells from the cell waste reservoir cell pools, under microscopic observation, when a single cell reaches the detection channel when the inlet, the cell pools and cell waste reservoir electrode suspension; buffer reservoir will run at the same time, a high voltage battery cell lysate, a waste reservoir is grounded; in that mode voltage, to protect cells, cell lysates, screening agents, parallel running buffer into the detection channel, and complete the separation of inclusion lysate of a single cell; connotation was detected at both ends of the detection channel; wherein 述细胞浓度应在103个/mL-106个/mL之间;单细胞细胞裂解液浓度应在0.01%-1%之间;筛分剂浓度应在0.15%-10%之间。 Said cell concentration should be between 103 / mL-106 th / mL; concentration of single cell lysis should be between 0.01% -1%; screening agent concentration should be between 0.15% and 10%.
  2. 2、 按照权利要求1所述基于微流控芯片的单细胞内涵物的分析方法, 其特征在于:细胞选用各种没有细胞壁的细胞。 2, according to the analysis method based on a single cell of inclusion microfluidic chip as claimed in claim wherein: the choice of a variety of cells without cell walls of the cells.
  3. 3、 按照权利要求1所述基于微流控芯片的单细胞内涵物的分析方法,其特征在于:细胞裂解液为十二烷基磺酸钠、十二烷基硫酸钠、十四烷基硫酸钠、十二烷基三甲基氯化铵、十二甲基三甲基溴化铵、聚氧化乙烯-23-十二垸基醚Brij35、辛基葡萄糖苷、TritonX-100之一种。 3, according to the analysis method based on a single cell of inclusion microfluidic chip as claimed in claim wherein: cell lysate of sodium dodecyl sulfate, sodium dodecyl sulfate, sodium tetradecyl sulfate sodium dodecyl trimethyl ammonium chloride, dodecyl methyl bromide, polyoxyethylene ether Brij 35 -23- twelve embankment, octyl glucoside A of TritonX-100.
  4. 4、 按照权利要求1所述基于微流控芯片的单细胞内涵物的分析方法, 其特征在于:筛分剂选自羟丙基甲基纤维素HPMC、羟乙基纤维素HEC、 聚丙烯酰胺PA、聚吡咯烷酮PVP、聚环氧乙烷PEO、聚乙烯醇PVA、甲基纤维素MC、聚糖之一种。 4. The analysis method based on the single cell 1 of inclusion microfluidic chip as claimed in claim wherein: HPMC screening agent is selected from hydroxypropyl methyl cellulose, hydroxyethyl cellulose HEC, polyacrylamide PA, polyvinylpyrrolidone PVP, polyethylene oxide PEO, polyvinyl alcohol PVA, methyl cellulose MC, of ​​a glycan.
  5. 5、 按照权利要求1所述基于微流控芯片的单细胞内涵物的分析方法, 其特征在于:所用微流控芯片材料是石英、玻璃、硅或者PMMA、 PDMS 聚合物。 5. The analysis method based on the single cell 1 of inclusion microfluidic chip as claimed in claim wherein: the microfluidic chip used material is quartz, glass, silicon, or PMMA, PDMS polymers.
  6. 6、 权利要求1〜5之一所述基于微流控芯片的单细胞内涵物的分析方法, 用于单细胞内核酸,蛋白质,氨基酸,糖等物质的分离分析。 6, any one of claims 1 ~ 5 Analysis of inclusion microfluidic chip based on a single cell, a single cell for separation and analysis of a nucleic acid species, proteins, amino acids, sugars and the like.
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CN103348215A (en) * 2010-11-07 2013-10-09 科学与工业研究理事会 On-chip 4D lightfield microscope
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CN103331097B (en) * 2013-05-27 2015-04-08 陕西师范大学 Application of polydimethylsiloxane micro fluidic chip in separating oligosaccharide and polysaccharide

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