CN101556273B - 利用电阻抗传感技术分析细胞迁移的方法及其专用装置 - Google Patents

利用电阻抗传感技术分析细胞迁移的方法及其专用装置 Download PDF

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CN101556273B
CN101556273B CN2008101035267A CN200810103526A CN101556273B CN 101556273 B CN101556273 B CN 101556273B CN 2008101035267 A CN2008101035267 A CN 2008101035267A CN 200810103526 A CN200810103526 A CN 200810103526A CN 101556273 B CN101556273 B CN 101556273B
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王磊
朱璟
邓橙
程京
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Tsinghua University
CapitalBio Corp
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Abstract

本发明公开了利用电阻抗传感技术分析细胞迁移的方法及其专用装置。该方法包括如下步骤：1)处理设有至少一对电极的绝缘基底，使所述电极上形成自组装单分子层，该单分子层能够阻止细胞在电极表面的贴附，所述电极上施加电信号后该分子层能够被解离；2)培养待检测细胞，使待检测细胞长满除覆盖有自组装单分子层的所述电极外的区域；3)在所述电极上施加电信号，使得电极上修饰的自组装单分子层解离；4)利用电阻抗传感方法对待测细胞在电极上的迁移情况进行检测。本发明还公开了分析细胞迁移的专用装置。该方法和装置具有实时性和定量性，可在药物筛选或评价中应用。

Description

利用电阻抗传感技术分析细胞迁移的方法及其专用装置
技术领域
本发明涉及一种分析细胞迁移的方法及其专用装置，特别涉及利用电阻抗传感技术分析细胞迁移的方法及其专用装置。 
背景技术
细胞迁移是一种细胞的运动现象，是细胞在化学信号或其他因素的影响下在空间上发生的位置转移。在包括胚胎发育、创伤愈合、免疫应答以及肿瘤转移在内的众多生理和病理过程中，细胞迁移都扮演着极为重要的角色。在肿瘤治疗过程中，肿瘤转移是导致患者死亡率增高的重要因素，而肿瘤转移则与肿瘤细胞的迁移有着极为密切的联系，为了攻克肿瘤，在抗肿瘤药物筛选和药物研发中人们也把目光投向了对肿瘤细胞迁移有抑制作用的抗转移类药物。这些都说明对于细胞迁移的分析无论对于生命科学、医学等多学科的基础研究还是对于药物工业等现代化生物技术领域来说都具有重要的意义。而传统的对细胞迁移进行检测的方法，例如愈伤分析，通常需要在体外培养的单层细胞上形成物理刮痕，然后利用显微镜对细胞迁移过程进行光学观察。这些方法往往依赖于繁重的手工劳动，而且实验重复性低，依赖于定性观察，难以进行定量测量。因而，难以满足现代生物学研究以及现代生物技术产业对细胞迁移测量的需要。这成为摆在诸多科研工作者面前的一个难题。 
自组装单分子层技术是一种利用某些分子的自组装特性在基底(例如玻璃、硅、金等)表面自动形成整齐排列的分子层结构的技术，依据用途以及基底类型的不同，有各种不同的单分子层。 
细胞电阻抗传感技术是一种能够对细胞的“形态学”变化进行实时、定量、无需标记以及无创伤检测的自动化电测量技术。这种技术的原理是在绝缘基底上加工微电极或微电极阵列，并在基底上进行细胞培养，当在电极上施加微弱的交流电信号时，由于细胞的绝缘性质，其会对电场造成一定的阻碍作用，通过对这种阻碍作用(阻抗)的测量，可以间接测量细胞的生物学行为。例如，当有细胞在培养环境中贴附、增殖或者其形态发生一定的变化时，其与基底之间的黏附状态发生变化时，其对电场的阻碍作用也会发生变化。通过对阻抗的测量就可以将细胞的这种变化动态地测量出来。细胞电阻抗传感技术已经被应用于进行细胞增殖(cell proliferation)， 细胞贴附(cell attachment)，细胞微运动(cell micromotion)，细胞屏障功能研究(cell barrier function)，细胞趋化现象研究(chemotactic cell motility)，细胞凋亡(cell apoptosis)，细胞愈伤分析(wound-healing assay)，体外细胞毒性分析(in vitrocell cytotoxicity)以及细胞对外界物理或化学刺激的响应(cell response to physicaland chemical changes)等相关研究。 
2004年Keese等报到了一种利用强烈细胞电穿孔形成没有细胞的空白区域，配合细胞电阻抗传感技术进行细胞迁移分析的方法(Keese et al.，2004)。在该方法中，金属电极被加工在用于细胞培养的绝缘基底上，将待分析的细胞接种于绝缘基底上并进行细胞培养，待细胞长满基底(当然，也长满电极)，在电极上施加幅值较大的电脉冲，使得电极区域的细胞由于强烈的不可逆细胞电穿孔而被杀死，造成电极区域出现没有细胞的空白区域，此时细胞开始由电极周围向电极表面区域迁移，用细胞电阻抗传感技术实时监测细胞在电极上的迁移状况，便可以检测细胞迁移进程。这种方法将细胞迁移的检测过程高度自动化地完成了，这是其优点，但是强烈的细胞电穿孔会对电极周围细胞的活性造成不良影响，另外，电极区域也会残留细胞死亡后留下的残骸，这些都有可能影响细胞迁移的检测结果。 
发明内容
本发明的目的是提供一种利用电阻抗传感技术分析细胞迁移的方法及其专用装置。该方法能够实现实时、连续测量细胞在自然培养状态下的迁移速率。 
本发明所提供的利用电阻抗传感技术分析细胞迁移的方法，包括如下步骤： 
1)处理设有至少一对电极的绝缘基底，使所述电极上形成自组装单分子层，该单分子层能够阻止细胞在电极表面的贴附，在所述电极上施加电信号后该分子层能够被解离； 
2)培养待检测细胞，使待检测细胞长满除覆盖有自组装单分子层的所述电极外的区域； 
3)在所述电极上施加电信号，使得电极上修饰的自组装单分子层解离； 
4)利用电阻抗传感方法对待测细胞在电极上的迁移情况进行检测。 
其中，所述电极为金属单电极和/或金属电极阵列；所述金属为金或铂；所述自组装单分子层由巯基化合物形成，如HS(CH2)11(OCH2CH2)6OH。所述电信号是直流信号、一种频率的交流信号或多种频率交流信号；所述待测细胞为肿瘤细胞。 
本发明的另一个目的是提供一种用所述方法分析细胞迁移的专用装置。 
本发明提供的用所述方法分析细胞迁移的专用装置，包括一个阻抗测量系统和至少一个细胞迁移组件，所述细胞迁移组件包括用于培养细胞的腔体、固设于所述腔体中的电极器件；所述电极器件包括绝缘基底和加工于所述绝缘基底上的电极；所述至少一个细胞迁移组件通过所述电极器件与所述一个阻抗测量系统电连接，其中，所述电极上自组装有单分子层，该单分子层能够阻止细胞在电极表面的贴附，所述电极上施加电信号后该分子层能够被解离。 
所述电极为金属单电极和/或金属电极阵列；所述金属为金或铂；所述自组装单分子层由聚乙醇巯基化合物形成，如HS(CH2)11(OCH2CH2)6OH；所述电信号是直流信号、一种频率的交流信号或多种频率交流信号；所述待测细胞为肿瘤细胞。 
本发明所述的方法、所述的装置可在药物筛选或药物评价中的应用。 
具体可按照如下方法进行： 
a、在所述装置的腔体中接种并培养待测细胞，加入待测化合物； 
b、测量细胞迁移的速率； 
c、评估待测化合物对待测细胞迁移速率的影响。 
本发明利用自组装技术在电极上形成致密的单分子层，阻止细胞在电极上的贴附和生长，最终形成没有细胞贴附的电极区域，当在电极上施加一定强度的电脉冲时，这种单分子层便会解离并离开电极，细胞从而可以从绝缘基底向电极迁移，当细胞在电极上迁移时可以阻碍电极之间的电流流动，引发测量的阻抗发生变化，这种阻抗的变化可以实时反映细胞迁移的进程，从而可以对细胞迁移过程进行实时、定量的分析。由于这种电阻抗测量的方法具有高通量和高度自动化的特征，具有实时性和定量性的优势，所以测量可以全自动进行，无需人工干预，并且具有易于在微型全分析系统中集成等诸多优势。 
附图说明
图1为用于细胞迁移自动化分析装置的示意图。其中，A为测量电极，B为参比电极，C为细胞培养腔体，D和E分别为用于将参比电极/测量电极与阻抗测量系统连接的端口，F为绝缘基底，G为阻抗测量系统。 
图2为用于实现高通量细胞迁移自动化分析装置的示意图。 
图3为利用细胞迁移分析装置分析鼠成纤维细胞系NIH/3T3细胞的迁移 
(A)为利用细胞迁移分析装置测得的阻抗曲线；(B)为在图(A)曲线的B点时拍摄的显微镜照片；(C)为在图(A)曲线的C点时拍摄的显微镜照片；(D)为 在图(A)曲线的D点时拍摄的显微镜照片；(E)为在图(A)曲线的E点时拍摄的显微镜照片。 
图4为利用细胞迁移分析装置分析秋水仙素抑制人宫颈癌CasKi细胞迁移 
(A)为利用细胞迁移分析装置测得的阻抗曲线；(B)为利用公式1计算得到的迁移的平均速率。 
具体实施方式
实施例1、制备用于细胞迁移分析的装置 
用于分析细胞迁移的装置的示意图如图1所示，包括用于培养细胞的腔体C、固设于所述腔体C中的电极器件和与所述电极器件电连接的阻抗测量系统G。所述电极器件由绝缘基底F以及加工在绝缘基底F上的微电极阵列A、单电极B、接点D和E构成。其中，微电极阵列A为测量电极，单电极B为参比电极。阻抗测量系统G通过接点D和E与电极器件相连。阻抗测量系统G为能够对电极之间阻抗的模数进行测量的仪器，例如安捷伦公司的阻抗分析仪(HP 4284A precision LCR meter)。 
该细胞迁移分析的装置可用如下方法制备：用微加工技术在绝缘基底上加工出金属电极阵列，将带有金属电极阵列的绝缘基底镶嵌于能够进行体外细胞培养的腔体底部，并使巯基化合物在电极上形成自组装单分子层，该单分子层能够阻止细胞在其表面的贴附。当在测量电极上施加特定的电信号(如用幅值为1.5V的直流电信号施加于测量电极上30秒；测量电极作为阴极，插入培养细胞腔体的铂丝作为阳极)后该单分子层能够被解离，最后将测量电极和参比电极与阻抗测量系统相连，构成完整的细胞迁移测量装置。具体的制备方法如下： 
首先，将用于加工细胞迁移分析装置的玻璃基片用浓硫酸/双氧水(体积比3∶1)清洗干净。然后，用微加工工艺中的光刻技术按照图1所示的电极结构进行光刻，形成图1中所示的电极图形。接下来，利用微加工工艺中的磁控溅射技术按照图1中电极结构的图案将金溅射在玻璃基片上，形成能够进行阻抗传感测量的微电极阵列，将带有微电极阵列的玻璃基片镶嵌于能够进行体外细胞培养的腔体底部，然后在细胞培养腔体中加入1.5mmol/L的HS(CH2)11(OCH2CH2)6OH(sigma-aldrich，675105)对微电极阵列进行浸泡，时间为6个小时，温度为室温，(HS(CH2)11(OCH2CH2)6OH与金电极表面结合而形成致密的自组装单分子层，这种单分子层能够阻止细胞的贴附。最后将金电极与阻抗测量系统相连，构成完整的细胞迁移分析装置。 
本实施例中应用的是一对电极，但也可以是多对电极，如二对、三对，电极之间可以组合，例如两对电极时，一共四个电极，可以两两连接，构成参比电极和测量电极。再如三对电极时，一共六个电极，可以任选三个电极相互连接构成测量电极，其余三连接后构成参比电极。 
本发明所述方法可以进行高通量测量，如附图2所示，阻抗测量系统与多个细胞迁移组件连接即得到高通量的细胞迁移分析的装置。 
实施例2、利用细胞迁移分析装置分析鼠成纤维细胞系NIH/3T3细胞的迁移 
在细胞迁移分析装置的细胞培养腔体中接种鼠成纤维细胞系NIH/3T3细胞，并在细胞培养箱内进行细胞培养，约24小时后，待细胞在绝缘基底上长满后，在电极上施加一个直流脉冲，金电极做负极，插入溶液的铂丝做正极，电压幅值为0.8V，时间约为30秒，单分子层可以从电极上去除。此时细胞开始向金电极上迁移，实时测量电极间的阻抗(用于阻抗测量所施加的交流电频率为40kHz，电极间的电压在微伏特量级)，随着细胞在电极上的迁移，阻抗信号逐渐变大，约6个小时后阻抗信号达到饱和值。细胞开始迁移到细胞长满整个电极所需要的时间T可以从阻抗曲线获得。细胞开始迁移到细胞长满整个电极所需要的时间T为从施加直流脉冲电信号开始到阻抗曲线饱和为止所需的时间。根据公式1对细胞迁移的平均速率(AS)进行计算。 
AS＝R×t-1(1) 
其中，R为测量电极(图1A)的半径。 
对HIH-3T3细胞的迁移进行分析的实验结果如图3所示。实验独立重复3次。其中图3(A)为利用细胞迁移分析装置测得的阻抗曲线；(B)为在图(A)曲线的B点时拍摄的显微镜照片，用自组装单分子层技术所形成的细胞图案，可以看出圆形的金电极上没有细胞贴附，而绝缘基底上已经布满了细胞，形成细胞单层。这有力地验证了本发明用自组装单分子层技术形成“创伤”的实验结果。(C)为在图(A)曲线的C点时拍摄的显微镜照片；(D)为在图(A)曲线的D点时拍摄的显微镜照片；(E)为在图(A)曲线的E点时拍摄的显微镜照片。这些显微镜照片验证了阻抗曲线测量的结果，即细胞在电极上迁移得越远，阻抗越大。在图3(A)中的E点所示位置，阻抗基本饱和，因而从图3(A)中的B点到E点的时间间隔即为NIH-3T3细胞开始迁移到细胞长满整个电极所需要的时间T(这里T约为6小时)，用电极的半径(200μm)和时间T根据公式1便可以计算细胞迁移的平均速率AS＝33.33μm/h。 
实施例3、利用细胞迁移分析装置分析秋水仙素抑制人宫颈癌CasKi细胞迁移 
在细胞迁移分析装置的细胞培养腔体中接种人宫颈癌CasKi细胞(ATCC，Manassas，VA)，并在细胞培养箱内进行细胞培养约24小时，待细胞在绝缘基底上长满后，在电极上施加一个直流脉冲，电极做负极，插入溶液的铂丝做正极，电压幅值约为0.8 V，时间约为30秒，单分子层可以从电极上去除，此时在细胞培养基中加入浓度分别为0.1μM、0.2μM和0.4μM的秋水仙素(sigma-aldrich，675105)，并进行阻抗测量。实验结果如图4所示。实验独立重复了3次。其中图4(A)为测得的阻抗曲线，可以明显观察到相比于对照(未加秋水仙素)，加入秋水仙素的细胞迁移速度明显减慢，并且秋水仙素浓度越高，抑制效果就越发明显，呈现出秋水仙素浓度依赖性的特征。图4(B)为根据公式1计算得到的迁移平均速率。 
上述实验结果表明本发明用自组装单分子层技术与细胞电阻抗传感技术相结合而制备的细胞迁移分析装置能够对细胞迁移过程进行定量、实时和自动化的分析。本发明的细胞迁移分析装置和方法能对待测化合物进行药物筛选和评价。 

Claims (9)

1.利用电阻抗传感技术分析细胞迁移的方法，包括如下步骤：
1）加工形成含有至少一对电极的绝缘基底，使所述电极上形成自组装单分子层，该单分子层能够阻止细胞在电极表面的贴附，所述电极上施加电信号后该分子层能够被解离；所述自组装单分子层由聚乙醇巯基化合物形成；
2）培养待检测细胞，使待检测细胞长满除覆盖有自组装单分子层的所述电极外的区域；
3）在所述电极上施加电信号，使得电极上修饰的自组装单分子层解离；
4）利用电阻抗传感方法对待测细胞在电极上的迁移情况进行检测；
所述聚乙醇巯基化合物为HS(CH2)11(OCH2CH2)6OH；
所述利用电阻抗传感技术分析细胞迁移的方法为非疾病诊断和治疗方法。
2.根据权利要求1所述的方法，其特征在于：所述电极为金属单电极或金属电极阵列。
3.根据权利要求2所述的方法，其特征在于：所述金属为金或铂。
4.根据权利要求1或2所述的方法，其特征在于：所述电信号是直流信号、一种频率的交流信号或多种频率交流信号。
5.一种用于细胞迁移分析的装置，包括一个阻抗测量系统和至少一个细胞迁移组件，所述细胞迁移组件包括用于培养细胞的腔体、固设于所述腔体中的电极器件；所述电极器件包括绝缘基底和加工于所述绝缘基底上的电极；所述至少一个细胞迁移组件通过所述电极器件与所述一个阻抗测量系统电连接，其特征在于：所述电极上自组装有单分子层，该单分子层能够阻止细胞在电极表面的贴附，所述电极上施加电信号后该分子层能够被解离；
所述自组装单分子层由聚乙醇巯基化合物形成；
所述聚乙醇巯基化合物为HS(CH2)11(OCH2CH2)6OH。
6.根据权利要求5所述的装置，其特征在于：所述电极为金属单电极或金属电极阵列。
7.根据权利要求6所述的装置，其特征在于：所述金属为金或铂；
8.根据权利要求5或6所述的装置，其特征在于：所述电信号是直流信号、一种频率的交流信号或多种频率交流信号。
9.权利要求1至4中任一所述的方法或权利要求5至8中任一所述的装置在药物筛选或药物评价中的应用。
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