电容细胞计数

1、电容细胞计数仪:单个生物细胞测定技术 编号:MFC-001L. L. Sohn*, O. A. Saleh*, G. R. Facer*, A. J. Beavis, R. S. Allan, and D. A. NottermanDepartments of *Physics and Molecular Biology, Princeton University, Princeton, NJ 08544Communicated by Lewis T. Williams, Chiron Technologies, Emeryville, CA, August 1, 2000 (received

2、 for review May 19, 2000) Abstract Measuring the DNA content of eukaryotic cells is a fundamental task in biology and medicine. We have observed a linear relationship between the DNA content of eukaryotic cells and the change in capacitance that is evoked by the passage of individual cells across a

3、1-kHz electric field. This relationship is species-independent;consequently, we have developed a microfluidic techniquecapacitance cytometrythat can be used to quantify the DNA content of single eukaryotic cells and to analyze the cell-cycle kinetics of populations of cells. Comparisons with standar

4、d flow cytometry demonstrate the sensitivity of this new technique.生物细胞的电子学特征具有重要的意义,它为人们提供了开发新的、快速的疾病诊断手段的机会,(1,2)以及供电子学研究的集成杂交芯片(3-7).以前对细胞的电子学研究主要集中在细胞的外部形态学研究,比如细胞膜的应答反应、细胞容积,这些研究初步得到了细胞的宏观装配信息(8,9).这里我们报道了一种创新的技术-集成微流控芯片.在这种芯片上,我们可以对单个细胞的一些内部特征进行观察.我们称此技术为“电容细胞计数仪”. 电容细胞计数仪可以对真核细胞内的DNA含量进行定量测定,

5、真核细胞包括从酵母到哺乳动物细胞等各种有机体.此外, 电容细胞计数仪可以用来测定细胞内DNA含量的异常变化,如细胞的癌变.通过监测细胞群DNA含量的变化,可以绘制细胞分化周期图.因此, 电容细胞计数仪在未来可以用作医疗诊断工具,具有极低的检测限,不必经过特殊处理即可检测到小量的组织样品中发生癌变的单个细胞的存在.尽管传统的激光流式细胞仪也可以对细胞逐个观察,但需要样品预处理,如染色或细胞进行处理. 相比之下,电容细胞计数仪不需要样品处理,并且操作更简单、快速、成本低.电容细胞计数仪的原理是基于对交变电流电容的测定.先进且灵敏的电子技术可以作到用探针对各种有机和无机材料以及外部电场的电极反应.电

6、容测定在过去主要用于对大量材料进行研究(10). 近年来,该技术被用于生物细胞装配研究,通过测定细胞大小和细胞膜电容,研究细胞分化过程(8)以及细胞分化成正常细胞和血液中的白细胞(9).与此相比,我们采用电容测定作为对单个真核细胞的细胞核内DNA的电极反应的测定并定量的方法.DNA是高度带电荷的分子,在低频交变电场中,同时与环境中和作用的结合,它的电极效应是相当大的(11-13). 测量这种电极反应作为总电容的变化,用CT,表示,用一对横挎的微电极表示逐个流经微流通道的缓冲溶液中的单个真核细胞.( Fig. 1所示). 不同于库尔特公司的流式细胞仪,它是测量流失的体积作为流经小孔的细胞的(14

7、).当细胞通过电场时,集成微流控芯片可测量细胞的电极反应.这些数据在一定程度上是和细胞内电荷分布相关的.Abbreviation: PDMS, polydimethyl siloxane. To whom reprint requests should be addressed at: Department of Physics, Jadwin Hall, Princeton University, Princeton, NJ 08544. E-mail: or .Smaller electrodes separ

8、ated by 10 nm and fabricated by using standard electron-beam lithography can be used in similar devices to detect single biomacromolecules (O.A.S. and L.L.S., unpublished results).The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereb

9、y marked “advertisement” in accordance with 18 U.S.C. 1734 solely to indicate this fact. Article published online before print: Proc. Natl. Acad. Sci. USA, 10.1073/pnas.200361297. Article and publication date are at /cgi/doi/10./.200361297PNAS / September 26, 2000 / vol. 97 /no. 20 /1068

10、710690(a) (b)Fig. 1. Schematic illustration of the integrated microfluidic device. (a) Top view shows the entire device, including electrode configuration, inlet and outlet holes for fluid, and the PDMS microfluidic channel. The electrodes are made of gold and are 50umwide.The distance,d, separating

11、 the electrodes is30um.The width of the PDMS microfluidic channel is also d, the length, L, is 5 um, and the height, h, is either30um or 40um.(b) Side view along the vertical axis of the device shows a detailed view of fluid delivery. Fluid delivery is accomplished with a syringe pump at nonpulsatil

12、e rates ranging from 1 to 300 ul/hr.Fig. 2. Device response over a course of 1,000ms to fixed mouse myeloma SP2/0 cells suspended in 75% ethanoly25% PBS solution at 10C. Distinct peaks are present in the data; each peak corresponds to a single cell flowing past the electrodes. The slight difference

13、in peak widths is an artifact of the time resolution limit of the data acquisition. The channel height of the device was 3um导电溶液中的电子测量结果因电极表面的电荷选择效应,结果复杂.我们的报道中,因这些离子效应使我们不能很好地解释电容的绝对值.但是,因为电极极性存在于电极表面,且对于特定形状装置,离子浓度,电源频率保持恒定,因此.当不同的细胞通过该装置时,我们可以通过比较总电容值的变化测定细胞的特征.集成微流控芯片装置的构造经过多步制成.采用照相平版印刷技术在玻璃或石英

14、底板上制成一对宽50um的微小金电极,构成感应器(Fig. 1a).两个独立电极间的距离是30um,即是真核细胞平均直径的3倍.然后在电极的两侧挖一个um级进液孔和一个排液孔. 再用柔和的光蚀刻技术(15)制作一个聚二甲基硅氧烷(PDMS)材料的微流通道.为了避免细胞直接通过一个电极所导致的复杂性以及为了使电极极性效应最小,将通道宽度作成两个电极间距离的大小.实验中,我们采用了两个不同的通道深度, h =30 um 和 h =40 um,发现h =40 um的灵敏度低于h =30 um,但定量结果相似.当将PDMS通道放置在电极和小孔上时( Fig. 1b),用微注射泵(KD2100型,KD

15、Scientific公司)输送液体到装置中,无脉冲,流速要求1ul/hr300ul/hr.我们用商品用的电容桥测量电容(AH2500A 1-kHz Ultra-Precision Capacitance Bridge, Andeen Hagerling, Cleveland).装置两端电桥的电压(Vrms =250 mV),频率=1kHz.通过电屏蔽整个装置以及精确控制温度(0.05C以内),可以达到:当通道干燥时,噪声水平约5 aF;当通道湿的状态时0.12 fF.我们可以用该装置来比较单个真核细胞的DNA含量.因为细胞周期中的不同阶段细胞的DNA含量不同. G0/G1 期的细胞有2倍的DN

16、A, G2/M期的细胞有4倍DNA, S期细胞的DNA含量介于2倍和4倍DNA之间. 我们还可以测定细胞所处的周期,带电荷的DNA的含量变化,导致细胞电容变化,变化的幅度使频率足以达到1 kHz.因此, G2/M期细胞总电容变化CT是G0/G1期细胞的2倍, S期细胞应介于G0/G1和G2/M之间,最终的超二倍体细胞(多于4倍DNA)电容应大于任何G0/G1 期,S期, 或G2/M期细胞的电容.AVrms=250mV is applied such that it represents a small perturbation to the entire system.At this volt

17、age, electrolysis and dielectrophoretic trapping can be ignored because both occur at higher voltages, Vrms . 1V. In addition, Vrms = 250 mV produces an optimal signal-to-noise ratio for the particular capacitance bridge used.iFor a movie of the cells flowing through the microfluidic channel, see ht

18、tp:/.实验中采用小鼠骨髓瘤细胞株(SP2/0), 一种癌变细胞株,在培养液中培养,使浓度达到105 个/ml.,用PBS溶液(pH 7.4)洗涤,再用75%乙醇在 220C 至少24 h固定,再用PBS溶液洗涤,再用Rnase酶处理,再洗涤,制成75% 乙醇储备液. 用核酸探针(SYTOX Green Nucleic Acid Stain;Molecular Probes)处理,然后用流式细胞仪分析 (FACScan flow cytometer;BectonDickinson Immunocytometry Systems),结果表明近 41%的细

19、胞处于 G0/G1 期, 40% 处于S 期, 18%处于 G2/M期,1% 处于超二倍体细胞期.在实验中,任何一次操作中ul级的细胞悬液(105 个/ml)以1ul/hr的流量注入到该装置中,用荧光探针(SYTOX Green Nucleic Acid Stain)标记的靶细胞,我们可以知道细胞逐个流经微通道的平均速度是250um/sec.雷诺尔德数(Reynolds number)估算为Re约1022, 因此证实了微通道中液流为层流状态.当细胞通过深度为30um 的通道时得到的电极反应如Fig. 2所示.图中得到了一系列尖峰, CT变化幅度从3 fF -12 fF. 各个峰间的时间间隔变化

20、为40 - 100 ms.测量过程中采用光学观察,证实了每个峰对应一个流经电极的单细胞.流式细胞仪的核心分析技术是DNA的矩形图.,它直观地以细胞数表示作为功能DNA的含量.,因此代表了细胞周期中处于各个时期的细胞比例.电容测量法的矩形图如Fig. 3所示.由图可知, SP2/0株存在两个不同的细胞群:对应于2倍DNA含量的峰,中心位于12.3 fF; 对应于4倍DNA含量的峰,中心位于23.0 fF.以次判断近48%的细胞处于G0/G1 期,30%处于 S期, 22%处于G2/M 期, 1%处于超二倍体细胞期. 这种分布是和由传统的流式细胞仪得到的结果一致的(Fig. 3 Inset).CT

21、 (rF)Fig. 3. Frequency histogram of the SP2/0 cells obtained with a device of h=30um, as compared with that obtained by conventional laser flow cytometry.The ungated histogram shows two major peaks, one centered at 12.3 fF,corresponding to G0/G1 phase, and one centered at 23.0 fF, corresponding to G

22、2/M phase. The distribution of cells at capacitances less than 10 fF correspond to hypodiploid cells; the distribution of cells at capacitances greater than 27 fF is due to hyperdiploid cells. Based on the histogram obtained, we judged that approximately 48% are in G0/G1 phase, 30% are in S phase, a

23、nd 22% are in G2/M phase. This cell cycle distribution is comparable to that obtained by conventional flow cytometry. (Inset) Histogram obtained via conventional flow cytometry. The data have been gated and do not include hypo- and hyperdiploid cells. Two peaks at fluorescence channels 190 and 380 c

24、orrespond to G0/G1 and G2/M phases, respectively.Fig. 3中的矩形图显著表明了我们的装置是可以区分处于不同时期的细胞.我们可以推断出测定的电容差是由于细胞流经通道时,不同的通道位置对电极的响应不同,因为我们已通过光学证实,细胞在通道的中心流过且直接流经两个电极之间.因为通道中的液流是层流状,无法观察细胞流经通道宽度时的边际形状.60多次装置经过测定得到相似结果,因此可以排除装置结构所产生的不确定性因素.实验证实,我们确实基于细胞的DNA含量而不是细胞的大小或容积来区分细胞.我们也测定并比较了鸟的血液红细胞(Accurate Scientifi

25、c,Westbury, NY)与羊的红细胞(Sigma)(细胞是用戊二醛固定的),然而鸟的红细胞含有2倍DNA,处于G0/G1 期,羊的红细胞与鸟的红细胞同样大小,但无DNA.当鸟红细胞流经装置时,测到电容峰,而羊的红细胞则无.多次实验得出同样结论.实验再次证明,我们确实是基于DNA的含量而不是细胞大小或容积来区分细胞的.鸟的红细胞具有平均电容变化CT,为5.0 fF.显而易见,鸟红细胞DNA含量少于SP2/0细胞,所以产生更小的信号.所观察到的不同种类细胞(5.012.3 fF)的信噪比与DNA含量具有不同寻常的定量一致性 2.5pg for Gallus domesticus vs. 6.

26、1 pg for Mus muscularis (16, 17).为了确定电容(CT)与DNA含量间的准确关系,我们对CT和不同细胞株DNA含量作图,结果如Fig. 4所示,在频率为1kHz.*时,二者间存在线性关系.这表明,在低频电场细胞流动产生的电容变化与DNA含量间的关系与物种无关.因为其它细胞器可能增大了DNA含量(如核酸组蛋白),我们不能断定全部的电容信号来源于DNA.但是CT与DNA含量间的关系满足我们的四个物种的细胞样品(酵母细胞、小鼠细胞、大鼠细胞、人细胞),因为这是不可能的,所有这些物种的DNA,其它核物质以及细胞质细胞器间具有相同的化学计量学关系.DNA含量与电容信号的线性

27、关系最可能的解释是,后者是前者的严格功能结果.DNA 含量( pg)Fig. 4. Change in capacitance, CT, vs. DNA content of mouse SP2/0, yeast,avian, and mammalian red blood cells. As shown, there is a linear relationship betweenCT, and DNA content at 1-kHz frequency. , Data taken with a device whose channel height was 30 um; , data t

28、aken with a device whose channel height was 40 um. The 40-um data were scaled by the ratio of theCT, values obtained for mouse SP2/0 cells measured with 30-um- and 40-um-high channel devices (see * footnote). All data were obtained at T=10C and in PBS solution.的确,通过电容细胞计数仪,可以检测DNA含量的过程变化.因此, 处于G0/G1

29、期大鼠-1(Rat-1)的粗大肌细胞放置在无血清培养基中(含 0.1% FBS) 培养72h,所有细胞是同步变化的,接着,这些细胞又被放入到含血清的培养基中(含 10% FBS).然后,收获等量的无血清培养基中的细胞和按一定时间间隔收获含血清培养基的细胞,用电容细胞计数仪(通道深度40um)和传统流式细胞仪测量DNA的变化.比较结果如Fig. 5,CT (fF) t=o h CT (fF) t=12hrs CT(fF) t=21hrsCT (fF) t=30hrs CT (fF) t=48hrsFig. 5. DNA progression of Rat-1, rodent fibroblas

30、t cells. Cells were G0/G1 arrested (t =0 h) and then allowed toprogress through one mitotic cell cycle in synchrony. At t = 12 h, the cells are beginning to enter S phase, and at t = 21 h, they have fully entered this phase. At t = 30 h, the cells have entered G2/M phase. This is shown by the second

31、ary peak atCTCT, CT, =6.4 fF. At t = 48 h, the cells have completed one mitotic cell cycle and are once again in G0/G1 phase. The G0/G1, S, and G2/M phases are indicated with arrows. The data shown were taken at T = 10C with a microfluidic channel whose height was 40um. Standard laserflowcytometry d

32、ata for the same population of cells are shown in the Insets for comparison表明培养在无血清培养基中的细胞(t = 0 h)是同步变化的,具有单一的CT值,中心位于3.2 fF,表明处于G0/G1期. 补加血清培养基后12 h,一些细胞的DNA含量已经升高,细胞处于S期,到培养21 h时,大部分细胞处于S期,DNA含量介于G0/G1和 G2/M期.30 h 时,许多细胞已经转化到G2/M期(中心位于 6.4 fF), 48 h时,细胞群又一次出现同步生长状态.总之,我们描述了一种测定单个真核细胞DNA含量的新技术-电容细

33、胞计数仪.DNA含量分析是研究细胞生理学的核心技术.这种技术具有常规流式细胞仪所不具有的优点,如操作简单,不需样品的特殊处理等.其应用十分广泛,如大量DNA含量测定,临床肿瘤样品中处于S期和非整倍体细胞的比例.此外还可以用于监测活体组织中DNA的含量,如外周血细胞、痰、脑脊液等.这种技术对肿瘤细胞检测具有极大优点并且可以实时监测药物剂量对细胞周期以及细胞调亡的效果.电容细胞计数仪也是唯一适合用于基于以微流控芯片技术的细胞分类研究,目前该领域的运用已制成了外部光纤检测器(18,19).*The ratio used to scale data taken with a 40-um-high ch

34、annel device was obtained by measuring mouse SP2/0 cells with both 30-um- and 40-um-high channel devices. The G0/G1 andG2/M peaks were centered at 3.75 fF and 7.50 fF, respectively, when cells were measured with a 40-um-high channel device; this, in contrast to the G0/G1 and G2/M peaks, centered at

35、12.3 fF and 23.0 fF, respectively, when the same cells were measured with a 30-um-high channel device.We thank J. D. Carbeck for advising on the microfluidics and T. E. Shenk and J. Broach for providing the rat and yeast cells, respectively. In addition, we thank R. Fitzgerald, E. Olson, and D. Weit

36、z for critical reading of this manuscript. O.A.S. acknowledges support from the. Fannie and John Hertz Foundation. This work was supported in part by National Science Foundation Grant DMR 96-24536, a DuPont Young.Faculty Award, and the NJ Commission on Science and Technology.1. Ayliffe, H. E., Frazi

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