電容細(xì)胞計(jì)數(shù)

1、電容細(xì)胞計(jì)數(shù)儀:單個(gè)生物細(xì)胞測(cè)定技術(shù) 編號(hào):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.生物細(xì)胞的電子學(xué)特征具有重要的意義,它為人們提供了開發(fā)新的、快速的疾病診斷手段的機(jī)會(huì),(1,2)以及供電子學(xué)研究的集成雜交芯片(3-7).以前對(duì)細(xì)胞的電子學(xué)研究主要集中在細(xì)胞的外部形態(tài)學(xué)研究,比如細(xì)胞膜的應(yīng)答反應(yīng)、細(xì)胞容積,這些研究初步得到了細(xì)胞的宏觀裝配信息(8,9).這里我們報(bào)道了一種創(chuàng)新的技術(shù)-集成微流控芯片.在這種芯片上,我們可以對(duì)單個(gè)細(xì)胞的一些內(nèi)部特征進(jìn)行觀察.我們稱此技術(shù)為“電容細(xì)胞計(jì)數(shù)儀”. 電容細(xì)胞計(jì)數(shù)儀可以對(duì)真核細(xì)胞內(nèi)的DNA含量進(jìn)行定量測(cè)定,

5、真核細(xì)胞包括從酵母到哺乳動(dòng)物細(xì)胞等各種有機(jī)體.此外, 電容細(xì)胞計(jì)數(shù)儀可以用來測(cè)定細(xì)胞內(nèi)DNA含量的異常變化,如細(xì)胞的癌變.通過監(jiān)測(cè)細(xì)胞群DNA含量的變化,可以繪制細(xì)胞分化周期圖.因此, 電容細(xì)胞計(jì)數(shù)儀在未來可以用作醫(yī)療診斷工具,具有極低的檢測(cè)限,不必經(jīng)過特殊處理即可檢測(cè)到小量的組織樣品中發(fā)生癌變的單個(gè)細(xì)胞的存在.盡管傳統(tǒng)的激光流式細(xì)胞儀也可以對(duì)細(xì)胞逐個(gè)觀察,但需要樣品預(yù)處理,如染色或細(xì)胞進(jìn)行處理. 相比之下,電容細(xì)胞計(jì)數(shù)儀不需要樣品處理,并且操作更簡(jiǎn)單、快速、成本低.電容細(xì)胞計(jì)數(shù)儀的原理是基于對(duì)交變電流電容的測(cè)定.先進(jìn)且靈敏的電子技術(shù)可以作到用探針對(duì)各種有機(jī)和無機(jī)材料以及外部電場(chǎng)的電極反應(yīng).電

6、容測(cè)定在過去主要用于對(duì)大量材料進(jìn)行研究(10). 近年來,該技術(shù)被用于生物細(xì)胞裝配研究,通過測(cè)定細(xì)胞大小和細(xì)胞膜電容,研究細(xì)胞分化過程(8)以及細(xì)胞分化成正常細(xì)胞和血液中的白細(xì)胞(9).與此相比,我們采用電容測(cè)定作為對(duì)單個(gè)真核細(xì)胞的細(xì)胞核內(nèi)DNA的電極反應(yīng)的測(cè)定并定量的方法.DNA是高度帶電荷的分子,在低頻交變電場(chǎng)中,同時(shí)與環(huán)境中和作用的結(jié)合,它的電極效應(yīng)是相當(dāng)大的(11-13). 測(cè)量這種電極反應(yīng)作為總電容的變化,用CT,表示,用一對(duì)橫挎的微電極表示逐個(gè)流經(jīng)微流通道的緩沖溶液中的單個(gè)真核細(xì)胞.( Fig. 1所示). 不同于庫爾特公司的流式細(xì)胞儀,它是測(cè)量流失的體積作為流經(jīng)小孔的細(xì)胞的(14

7、).當(dāng)細(xì)胞通過電場(chǎng)時(shí),集成微流控芯片可測(cè)量細(xì)胞的電極反應(yīng).這些數(shù)據(jù)在一定程度上是和細(xì)胞內(nèi)電荷分布相關(guān)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導(dǎo)電溶液中的電子測(cè)量結(jié)果因電極表面的電荷選擇效應(yīng),結(jié)果復(fù)雜.我們的報(bào)道中,因這些離子效應(yīng)使我們不能很好地解釋電容的絕對(duì)值.但是,因?yàn)殡姌O極性存在于電極表面,且對(duì)于特定形狀裝置,離子濃度,電源頻率保持恒定,因此.當(dāng)不同的細(xì)胞通過該裝置時(shí),我們可以通過比較總電容值的變化測(cè)定細(xì)胞的特征.集成微流控芯片裝置的構(gòu)造經(jīng)過多步制成.采用照相平版印刷技術(shù)在玻璃或石英

14、底板上制成一對(duì)寬50um的微小金電極,構(gòu)成感應(yīng)器(Fig. 1a).兩個(gè)獨(dú)立電極間的距離是30um,即是真核細(xì)胞平均直徑的3倍.然后在電極的兩側(cè)挖一個(gè)um級(jí)進(jìn)液孔和一個(gè)排液孔. 再用柔和的光蝕刻技術(shù)(15)制作一個(gè)聚二甲基硅氧烷(PDMS)材料的微流通道.為了避免細(xì)胞直接通過一個(gè)電極所導(dǎo)致的復(fù)雜性以及為了使電極極性效應(yīng)最小,將通道寬度作成兩個(gè)電極間距離的大小.實(shí)驗(yàn)中,我們采用了兩個(gè)不同的通道深度, h =30 um 和 h =40 um,發(fā)現(xiàn)h =40 um的靈敏度低于h =30 um,但定量結(jié)果相似.當(dāng)將PDMS通道放置在電極和小孔上時(shí)( Fig. 1b),用微注射泵(KD2100型,KD

15、Scientific公司)輸送液體到裝置中,無脈沖,流速要求1ul/hr300ul/hr.我們用商品用的電容橋測(cè)量電容(AH2500A 1-kHz Ultra-Precision Capacitance Bridge, Andeen Hagerling, Cleveland).裝置兩端電橋的電壓(Vrms =250 mV),頻率=1kHz.通過電屏蔽整個(gè)裝置以及精確控制溫度(0.05C以內(nèi)),可以達(dá)到:當(dāng)通道干燥時(shí),噪聲水平約5 aF;當(dāng)通道濕的狀態(tài)時(shí)0.12 fF.我們可以用該裝置來比較單個(gè)真核細(xì)胞的DNA含量.因?yàn)榧?xì)胞周期中的不同階段細(xì)胞的DNA含量不同. G0/G1 期的細(xì)胞有2倍的DN

16、A, G2/M期的細(xì)胞有4倍DNA, S期細(xì)胞的DNA含量介于2倍和4倍DNA之間. 我們還可以測(cè)定細(xì)胞所處的周期,帶電荷的DNA的含量變化,導(dǎo)致細(xì)胞電容變化,變化的幅度使頻率足以達(dá)到1 kHz.因此, G2/M期細(xì)胞總電容變化CT是G0/G1期細(xì)胞的2倍, S期細(xì)胞應(yīng)介于G0/G1和G2/M之間,最終的超二倍體細(xì)胞(多于4倍DNA)電容應(yīng)大于任何G0/G1 期,S期, 或G2/M期細(xì)胞的電容.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:/.實(shí)驗(yàn)中采用小鼠骨髓瘤細(xì)胞株(SP2/0), 一種癌變細(xì)胞株,在培養(yǎng)液中培養(yǎng),使?jié)舛冗_(dá)到105 個(gè)/ml.,用PBS溶液(pH 7.4)洗滌,再用75%乙醇在 220C 至少24 h固定,再用PBS溶液洗滌,再用Rnase酶處理,再洗滌,制成75% 乙醇儲(chǔ)備液. 用核酸探針(SYTOX Green Nucleic Acid Stain;Molecular Probes)處理,然后用流式細(xì)胞儀分析 (FACScan flow cytometer;BectonDickinson Immunocytometry Systems),結(jié)果表明近 41%的細(xì)

19、胞處于 G0/G1 期, 40% 處于S 期, 18%處于 G2/M期,1% 處于超二倍體細(xì)胞期.在實(shí)驗(yàn)中,任何一次操作中ul級(jí)的細(xì)胞懸液(105 個(gè)/ml)以1ul/hr的流量注入到該裝置中,用熒光探針(SYTOX Green Nucleic Acid Stain)標(biāo)記的靶細(xì)胞,我們可以知道細(xì)胞逐個(gè)流經(jīng)微通道的平均速度是250um/sec.雷諾爾德數(shù)(Reynolds number)估算為Re約1022, 因此證實(shí)了微通道中液流為層流狀態(tài).當(dāng)細(xì)胞通過深度為30um 的通道時(shí)得到的電極反應(yīng)如Fig. 2所示.圖中得到了一系列尖峰, CT變化幅度從3 fF -12 fF. 各個(gè)峰間的時(shí)間間隔變化

20、為40 - 100 ms.測(cè)量過程中采用光學(xué)觀察,證實(shí)了每個(gè)峰對(duì)應(yīng)一個(gè)流經(jīng)電極的單細(xì)胞.流式細(xì)胞儀的核心分析技術(shù)是DNA的矩形圖.,它直觀地以細(xì)胞數(shù)表示作為功能DNA的含量.,因此代表了細(xì)胞周期中處于各個(gè)時(shí)期的細(xì)胞比例.電容測(cè)量法的矩形圖如Fig. 3所示.由圖可知, SP2/0株存在兩個(gè)不同的細(xì)胞群:對(duì)應(yīng)于2倍DNA含量的峰,中心位于12.3 fF; 對(duì)應(yīng)于4倍DNA含量的峰,中心位于23.0 fF.以次判斷近48%的細(xì)胞處于G0/G1 期,30%處于 S期, 22%處于G2/M 期, 1%處于超二倍體細(xì)胞期. 這種分布是和由傳統(tǒng)的流式細(xì)胞儀得到的結(jié)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中的矩形圖顯著表明了我們的裝置是可以區(qū)分處于不同時(shí)期的細(xì)胞.我們可以推斷出測(cè)定的電容差是由于細(xì)胞流經(jīng)通道時(shí),不同的通道位置對(duì)電極的響應(yīng)不同,因?yàn)槲覀円淹ㄟ^光學(xué)證實(shí),細(xì)胞在通道的中心流過且直接流經(jīng)兩個(gè)電極之間.因?yàn)橥ǖ乐械囊毫魇菍恿鳡?無法觀察細(xì)胞流經(jīng)通道寬度時(shí)的邊際形狀.60多次裝置經(jīng)過測(cè)定得到相似結(jié)果,因此可以排除裝置結(jié)構(gòu)所產(chǎn)生的不確定性因素.實(shí)驗(yàn)證實(shí),我們確實(shí)基于細(xì)胞的DNA含量而不是細(xì)胞的大小或容積來區(qū)分細(xì)胞.我們也測(cè)定并比較了鳥的血液紅細(xì)胞(Accurate Scientifi

25、c,Westbury, NY)與羊的紅細(xì)胞(Sigma)(細(xì)胞是用戊二醛固定的),然而鳥的紅細(xì)胞含有2倍DNA,處于G0/G1 期,羊的紅細(xì)胞與鳥的紅細(xì)胞同樣大小,但無DNA.當(dāng)鳥紅細(xì)胞流經(jīng)裝置時(shí),測(cè)到電容峰,而羊的紅細(xì)胞則無.多次實(shí)驗(yàn)得出同樣結(jié)論.實(shí)驗(yàn)再次證明,我們確實(shí)是基于DNA的含量而不是細(xì)胞大小或容積來區(qū)分細(xì)胞的.鳥的紅細(xì)胞具有平均電容變化CT,為5.0 fF.顯而易見,鳥紅細(xì)胞DNA含量少于SP2/0細(xì)胞,所以產(chǎn)生更小的信號(hào).所觀察到的不同種類細(xì)胞(5.012.3 fF)的信噪比與DNA含量具有不同尋常的定量一致性 2.5pg for Gallus domesticus vs. 6.

26、1 pg for Mus muscularis (16, 17).為了確定電容(CT)與DNA含量間的準(zhǔn)確關(guān)系,我們對(duì)CT和不同細(xì)胞株DNA含量作圖,結(jié)果如Fig. 4所示,在頻率為1kHz.*時(shí),二者間存在線性關(guān)系.這表明,在低頻電場(chǎng)細(xì)胞流動(dòng)產(chǎn)生的電容變化與DNA含量間的關(guān)系與物種無關(guān).因?yàn)槠渌?xì)胞器可能增大了DNA含量(如核酸組蛋白),我們不能斷定全部的電容信號(hào)來源于DNA.但是CT與DNA含量間的關(guān)系滿足我們的四個(gè)物種的細(xì)胞樣品(酵母細(xì)胞、小鼠細(xì)胞、大鼠細(xì)胞、人細(xì)胞),因?yàn)檫@是不可能的,所有這些物種的DNA,其它核物質(zhì)以及細(xì)胞質(zhì)細(xì)胞器間具有相同的化學(xué)計(jì)量學(xué)關(guān)系.DNA含量與電容信號(hào)的線性

27、關(guān)系最可能的解釋是,后者是前者的嚴(yán)格功能結(jié)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.的確,通過電容細(xì)胞計(jì)數(shù)儀,可以檢測(cè)DNA含量的過程變化.因此, 處于G0/G1

29、期大鼠-1(Rat-1)的粗大肌細(xì)胞放置在無血清培養(yǎng)基中(含 0.1% FBS) 培養(yǎng)72h,所有細(xì)胞是同步變化的,接著,這些細(xì)胞又被放入到含血清的培養(yǎng)基中(含 10% FBS).然后,收獲等量的無血清培養(yǎng)基中的細(xì)胞和按一定時(shí)間間隔收獲含血清培養(yǎng)基的細(xì)胞,用電容細(xì)胞計(jì)數(shù)儀(通道深度40um)和傳統(tǒng)流式細(xì)胞儀測(cè)量DNA的變化.比較結(jié)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表明培養(yǎng)在無血清培養(yǎng)基中的細(xì)胞(t = 0 h)是同步變化的,具有單一的CT值,中心位于3.2 fF,表明處于G0/G1期. 補(bǔ)加血清培養(yǎng)基后12 h,一些細(xì)胞的DNA含量已經(jīng)升高,細(xì)胞處于S期,到培養(yǎng)21 h時(shí),大部分細(xì)胞處于S期,DNA含量介于G0/G1和 G2/M期.30 h 時(shí),許多細(xì)胞已經(jīng)轉(zhuǎn)化到G2/M期(中心位于 6.4 fF), 48 h時(shí),細(xì)胞群又一次出現(xiàn)同步生長(zhǎng)狀態(tài).總之,我們描述了一種測(cè)定單個(gè)真核細(xì)胞DNA含量的新技術(shù)-電容細(xì)

33、胞計(jì)數(shù)儀.DNA含量分析是研究細(xì)胞生理學(xué)的核心技術(shù).這種技術(shù)具有常規(guī)流式細(xì)胞儀所不具有的優(yōu)點(diǎn),如操作簡(jiǎn)單,不需樣品的特殊處理等.其應(yīng)用十分廣泛,如大量DNA含量測(cè)定,臨床腫瘤樣品中處于S期和非整倍體細(xì)胞的比例.此外還可以用于監(jiān)測(cè)活體組織中DNA的含量,如外周血細(xì)胞、痰、腦脊液等.這種技術(shù)對(duì)腫瘤細(xì)胞檢測(cè)具有極大優(yōu)點(diǎn)并且可以實(shí)時(shí)監(jiān)測(cè)藥物劑量對(duì)細(xì)胞周期以及細(xì)胞調(diào)亡的效果.電容細(xì)胞計(jì)數(shù)儀也是唯一適合用于基于以微流控芯片技術(shù)的細(xì)胞分類研究,目前該領(lǐng)域的運(yùn)用已制成了外部光纖檢測(cè)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

37、er, A. B. & Rabbitt, R. D. (1999) IEEE J. Microelectromechanical Syst. 8, 5057.2. Huang, Y., Yang, J., Wang, X. B., Becker, F. F. & Gascoyne, P. R. C. (2000) J. Hematother. Stem Cell Res. 8, 481490.3. Fromherz, P., Kiessling, V., Kottig, K. & Zeck, G. (1999) Appl. Phys. A 69, 571576.4. Vassanelli, S

38、. & Fromherz, P. (1997) Appl. Phys. A 65, 8588.5. Maher, M. P., Pine, J., Wright, J. & Yu-Chong, Tai. (1999) J. Neurosci. Methods 87, 4556.6. Kawana, A. (1996) in Nanofabrication and Biosystems, eds. Hoch, H. C.,Jelinski, L. W. & Craighead, H. G. (Cambridge Univ. Press, Cambridge, U.K.), pp. 258275 and references therein.7. Jung, D. R.,