阻抗在細(xì)胞檢測中的應(yīng)用進(jìn)展

胡清清，崔 垚，張 帆，何品剛

（華東師范大學(xué)化學(xué)與分子工程學(xué)院，上海200241）

阻抗在細(xì)胞檢測中的應(yīng)用進(jìn)展

胡清清，崔 垚，張 帆*，何品剛*

（華東師范大學(xué)化學(xué)與分子工程學(xué)院，上海200241）

細(xì)胞阻抗傳感技術(shù)是細(xì)胞培養(yǎng)和阻抗技術(shù)的結(jié)合，將細(xì)胞培養(yǎng)在工作電極上，通過施加微小的電壓，收集流經(jīng)包括細(xì)胞在內(nèi)的整個系統(tǒng)的電流信號來檢測細(xì)胞的狀態(tài)。阻抗技術(shù)由于實時、連續(xù)、不破壞性、無需標(biāo)記等優(yōu)點，已被廣泛應(yīng)用于細(xì)胞性質(zhì)測定、癌細(xì)胞檢測和抗癌藥物篩選等方面。該文綜述了阻抗技術(shù)在細(xì)胞檢測中的應(yīng)用和進(jìn)展，包括：1、阻抗檢測電極的設(shè)計；2、阻抗監(jiān)測細(xì)胞生理狀態(tài)；3、阻抗在細(xì)胞毒性和藥物篩選中的應(yīng)用；4、阻抗區(qū)分正常細(xì)胞與癌細(xì)胞。

阻抗；細(xì)胞檢測；綜述

0 引言

阻抗技術(shù)是一種以較小振幅正弦波電勢 (電流)作為擾動信號，使電極系統(tǒng)產(chǎn)生近似于線性關(guān)系響應(yīng)、測量頻率很寬的阻抗值。由于電路中的物質(zhì)對電流的阻礙作用不同，導(dǎo)致檢測到的阻抗值不同，從而反映物質(zhì)的相關(guān)性質(zhì)。

細(xì)胞阻抗傳感技術(shù)是將細(xì)胞培養(yǎng)和阻抗技術(shù)相結(jié)合，可在細(xì)胞培養(yǎng)的過程中對細(xì)胞的狀態(tài)進(jìn)行實時、連續(xù)地監(jiān)測，能夠及早發(fā)現(xiàn)細(xì)胞狀態(tài)的變化，被認(rèn)為是定量研究細(xì)胞行為最有前景的技術(shù)之一[1-2]。利用阻抗技術(shù)檢測細(xì)胞是將細(xì)胞培養(yǎng)在工作電極上，通過施加微小的電壓，收集流經(jīng)包括細(xì)胞在內(nèi)的整個系統(tǒng)的電流信號來檢測細(xì)胞的狀態(tài)。細(xì)胞被磷脂雙分子層構(gòu)成的細(xì)胞膜包裹著，使得細(xì)胞成為電的不良導(dǎo)體，直流電會被旁路，當(dāng)施加一定頻率的交流電時，電流流經(jīng)細(xì)胞就會受到一定阻礙，從而引起整個體系阻抗的變化。這種阻礙與細(xì)胞的性質(zhì)和狀態(tài)密切相關(guān)，通過收集到的電流信號就可以分析細(xì)胞信息。由于施加的電壓非常小，電流非常微弱，不會對細(xì)胞造成損傷，所以阻抗檢測可以貫穿細(xì)胞培養(yǎng)的整個過程[3-7]。細(xì)胞阻抗傳感技術(shù)由Giaever和Keese于1984年建立，他們將哺乳動物的成纖

維細(xì)胞（fibroblasts）培養(yǎng)在培養(yǎng)皿中，培養(yǎng)皿底部有兩個共面的金電極，細(xì)胞在工作電極上生長，當(dāng)施加4 kHz的正弦交流電場時，通過鎖相放大器獲得流經(jīng)細(xì)胞的電流信號，實驗結(jié)果表明粘附在電極上的細(xì)胞對電流有明顯的阻礙作用，并且隨時間發(fā)生變化[8]。該文綜述了阻抗技術(shù)近年來在細(xì)胞檢測中的應(yīng)用進(jìn)展。

1 阻抗檢測電極的設(shè)計

利用阻抗技術(shù)進(jìn)行細(xì)胞檢測，第一步是要將細(xì)胞培養(yǎng)和阻抗檢測結(jié)合于一體，這就涉及到阻抗檢測裝置的構(gòu)建，其中最關(guān)鍵的是培養(yǎng)基底要導(dǎo)電，通常是將細(xì)胞培養(yǎng)在導(dǎo)電的工作電極上，由培養(yǎng)液連接對電極，進(jìn)行檢測。根據(jù)工作電極數(shù)目的不同，阻抗檢測裝置可以分為單電極體系和陣列電極體系。

1.1 單電極

單電極是指阻抗檢測體系只含有一個工作電極和一個對電極，如圖1。通常情況下，選用導(dǎo)電性好的、面積小的電極作為工作電極，面積大的電極作為對電極，兩個電極的面積比值往往小于0.01，這就保證了體系阻抗的變化主要取決于工作電極表面的改變[9]。單電極系統(tǒng)應(yīng)用廣泛[10-14]，工作電極材料可選用金、鉑、銦錫氧化物（ITO）等，而對電極則多采用鉑。

圖1 單電極阻抗檢測體系Fig.1 ECIS with mono work electrode

1.2 陣列電極

陣列電極體系包含多個工作電極，其中可分為叉指電極和普通陣列電極。

1.2.1 叉指電極

叉指電極是陣列電極的一種特殊構(gòu)型，是指具有梳狀的、面內(nèi)有周期性圖案的電極，由多組并列的條形電極組成，其特點是由一個共同的末端相連接，形成類似手指交叉的電極陣列結(jié)構(gòu)。在叉指電極中，并列的條形電極大小一樣，材質(zhì)相同，因此電極的阻抗在總阻抗中所占的比例相等，不區(qū)分工作電極和對電極[15-17]，如圖2。叉指電極易于微型化，但是無法進(jìn)行單細(xì)胞分析。

圖2 叉指電極示意圖[17]Fig.2 Sketch map showing the layout of the ECIS sensor electrodes

1.2.2 普通陣列電極

普通陣列電極體系含有多個通道，每一個通道含一個工作電極，這些工作電極是相互獨立的，共用一個對電極，即每一個獨立的電路都含一套完整的工作電極和對電極，利用開關(guān)可在工作電極間切換。Applied Biophysics（Troy，NY）公司采用光刻等技術(shù)，在基底上形成了由8個獨立的工作電極（0.057 mm2）和1個共用的對電極（7×46 mm2）組成的阻抗檢測體系，可進(jìn)行多通道的同時測量[18-24]，如圖3所示。

圖3 陣列電極系統(tǒng)示意圖[20]Fig.3 Schematic diagram of the ECIS system

2 阻抗監(jiān)測細(xì)胞生理狀態(tài)

細(xì)胞阻抗技術(shù)可以實時、原位、長時間地反映電極表面細(xì)胞發(fā)生的變化，因此被廣泛應(yīng)用于各種細(xì)胞研究，包括細(xì)胞粘附和鋪展[25-33]、細(xì)胞

繁殖[34-38]、細(xì)胞遷移與修復(fù)[39-43]等。

2.1 粘附和鋪展

不同種類貼壁性細(xì)胞在電極表面的粘附能力不同，粘附所需時間也不一樣，不同的電極對細(xì)胞粘附的影響也有差異。對于不易粘附的電極，需要在電極表面附著一層粘附蛋白。Wegener等運用細(xì)胞阻抗技術(shù)檢測MDCK細(xì)胞在不同蛋白處理過的金電極表面的貼附情況，在他們的研究中提到，MDCK細(xì)胞在涂有粘附蛋白的電極表面更容易貼壁，并提出高頻電容是反映MDCK細(xì)胞早期粘附和鋪展最靈敏的參數(shù)[26]。Liu等利用多通道微陣列電極測定了細(xì)胞粘附和細(xì)胞形態(tài)的變化，他們將人類食道癌細(xì)胞（KYSE 30）培養(yǎng)在涂有粘連蛋白的電極表面，通過細(xì)胞粘附和鋪展引起的形態(tài)改變均可將阻抗值的變化體現(xiàn)出來，如圖4，細(xì)胞分別粘附在纖連蛋白預(yù)處理的和未處理的電極表面，當(dāng)細(xì)胞粘附在電極表面時，阻抗值有增加[27]。Asphahani等在金電極表面進(jìn)行小鼠成纖維細(xì)胞的圖案化，細(xì)胞通過lysinearginine-glycine-aspartic acid(KRGD)粘附肽固定在電極表面，這種粘附機(jī)理依賴于共價結(jié)合和物理吸附，結(jié)果表明單細(xì)胞圖案化能夠改進(jìn)細(xì)胞傳感器的阻抗特性[30]。對于細(xì)胞粘附而導(dǎo)致的阻抗變化，Giaever和Keese認(rèn)為有細(xì)胞時比沒細(xì)胞時體系阻抗之所以增大，是因為細(xì)胞粘附到電極表面后，導(dǎo)致空白電極面積減少，電子傳遞受阻，表現(xiàn)為阻抗增加[33]。

圖4 微電極陣列單個通道中癌細(xì)胞分別在有無纖連蛋白時粘附的阻抗值隨時間的變化曲線，檢測頻率為1 kHz[27]Fig.4 One channel impedance detection of the microelectrode array for cancer cells adhesion.Time course of the impedance magnitude at a sampling frequency of 1 kHz for fibronectin(FN)modified and non-modified electrode surfaces

2.2 細(xì)胞繁殖

細(xì)胞在電極上繁殖會導(dǎo)致阻抗增大，因此可根據(jù)阻抗的變化間接檢測細(xì)胞數(shù)目的變化[34]。Shih等利用自制的數(shù)字微流控系統(tǒng)對HeLa、NIH-3T3、CHO-K1等三種細(xì)胞進(jìn)行了4天的阻抗檢測，分析得到了細(xì)胞的繁殖速率，這種方法使分析試劑用量減少近1000倍[35]。Brischwein等將小鼠成纖維細(xì)胞（L929）培養(yǎng)在阻抗傳感器上，檢測細(xì)胞引起的阻抗改變，結(jié)果表明，細(xì)胞增殖導(dǎo)致阻抗顯著增大，在一定培養(yǎng)時間內(nèi)，阻抗隨時間逐漸增大[37]。

2.3 細(xì)胞遷移與修復(fù)

Keese等將細(xì)胞培養(yǎng)在金薄膜電極表面，正常模式下，1 μA，4 kHz的交流阻抗測試電流不會對細(xì)胞造成影響，當(dāng)施加2.5 V，40 kHz高壓脈沖時，細(xì)胞產(chǎn)生控制性損害，從電極上脫落，引起阻抗的下降，然后隨著細(xì)胞的遷移和修復(fù)，阻抗又恢復(fù)到融匯時的水平，文章優(yōu)化了脈沖作用時間、電極面積和細(xì)胞的種類對損傷再生測試的影響[39]。Wang等通過自組裝單層膜（selfassembled monolayers，SAMs）的脫落以使得細(xì)胞脫離電極表面，模擬細(xì)胞損傷，并對比了不同細(xì)胞的遷移差異[40]。Stolwijk等利用原位電穿孔技術(shù) （In situ electroporation，ISE）對細(xì)胞進(jìn)行損傷后，再施加藥物，研究細(xì)胞修復(fù)程度。當(dāng)施加損傷信號時，細(xì)胞膜被電穿孔破壞后導(dǎo)致細(xì)胞死亡。損傷電流施加時間不同會引起細(xì)胞損傷程度以及細(xì)胞再生過程的差異，這些均可通過細(xì)胞不同時期的阻抗值反映出來[41]。

3 阻抗在細(xì)胞毒性和藥物篩選中的應(yīng)用

細(xì)胞的生長過程是動態(tài)的，并且對周圍環(huán)境十分敏感，尤其是毒物。阻抗技術(shù)可做到實時表征細(xì)胞對周圍環(huán)境的反應(yīng)，細(xì)胞培養(yǎng)在電極上，體系中加入毒物作用以后，阻抗值下降，且毒物濃度越高，作用時間越長，阻抗值下降越明顯。根據(jù)藥物作用對象的不同，研究可分為兩類：毒物對正常細(xì)胞的毒性[44-47]和抗癌藥物對癌細(xì)胞的

作用[48-54]。

3.1 毒物對正常細(xì)胞的毒性

通過傳統(tǒng)的生物化學(xué)測定方法，如基于細(xì)胞膜通透性改變的臺盼藍(lán)染色法[55-56]、對細(xì)胞核染色的碘化丙啶法[57]、測定細(xì)胞酶活性的 MTT法[58-61]、外加標(biāo)記物等[62]，確定細(xì)胞的存活率，通常會破壞細(xì)胞的連續(xù)培養(yǎng)，難以實現(xiàn)實時分析。阻抗技術(shù)則突破了這些技術(shù)的局限，能夠在細(xì)胞的培養(yǎng)過程中，進(jìn)行動態(tài)觀察和原位檢測，記錄毒物作用的整個過程。Xiao等將氯化鎘（CdCl2），苯扎氯銨（BAK），砷酸鈉（Na2HAsO4）三種物質(zhì)作用于中國倉鼠肺細(xì)胞（V79），利用阻抗技術(shù)測定這些物質(zhì)對細(xì)胞的毒性，如圖5，當(dāng)施加毒性物質(zhì)作用后，細(xì)胞的阻抗值明顯下降，其結(jié)果與中性紅標(biāo)準(zhǔn)方法的結(jié)果一致[44]。這個課題組又將上述三種物質(zhì)以及氯化汞（HgCl2）和三硝基苯（TNB）作用于V79，前四種物質(zhì)作為急性毒物的代表，而TNB作為長期慢性毒物的代表，利用阻抗技術(shù)檢測毒物濃度和作用時間對V79細(xì)胞的影響，為細(xì)胞對毒物的動態(tài)反應(yīng)提供了信息[45]。其他課題組也做了很多相關(guān)研究，如用阻抗方法評估鎘[46]，Triton X-100[47]對細(xì)胞的影響等。

圖5 三種毒物（氯化鎘、苯扎氯銨和砷酸鈉）對成纖維V79細(xì)胞的影響[44]Fig.5 Responses(Ω per cell)of fibroblastic V79 cells to three cytotoxic chemicals(μmol/L):cadmium chloride,(a) 2.9,(b)4.6,(c)6.2,and(d)8.1;benzalkonium chloride,(a) 15.2,(b)18.3,(c)21.3,and(d)30.4;sodium arsenate,(a) 45,(b)60,(c)140,and(d)200

3.2 抗癌藥物對癌細(xì)胞的作用

許多抗癌藥物通過誘導(dǎo)細(xì)胞凋亡來殺死癌細(xì)胞。對細(xì)胞死亡的可靠評估，在發(fā)展有效、安全的抗癌藥物治療中顯得尤為重要。細(xì)胞凋亡過程中，細(xì)胞形態(tài)、細(xì)胞間的連接等發(fā)生改變，而細(xì)胞阻抗傳感技術(shù)能夠?qū)崟r檢測這些變化，降低了檢測成本，加快了測定流程，為抗癌藥物篩選提供了一種新的評估方法。清華大學(xué)周玉祥課題組通過細(xì)胞阻抗技術(shù)檢測阿司匹林誘導(dǎo)人結(jié)腸腺癌細(xì)胞（HT29）的改變，同時，通過透射電子顯微鏡成像佐證細(xì)胞形態(tài)改變，闡明了細(xì)胞阻抗技術(shù)在藥物毒性測試和藥物研發(fā)中的重要作用[48]。浙江大學(xué)王平課題組研究了癌癥治療的常用化學(xué)藥物-順鉑對人食管鱗癌細(xì)胞（KYSE30）的作用，細(xì)胞阻抗技術(shù)用于監(jiān)測細(xì)胞生長行為并探討了抗癌藥物的化學(xué)選擇性[27]。Pradhan等報導(dǎo)了利用阻抗技術(shù)評估抗癌藥物ZD6474對乳腺癌細(xì)胞（T47D、MCF-7）的作用，優(yōu)化了電極尺寸和測試頻率，測試結(jié)果與傳統(tǒng)的流式細(xì)胞儀測定結(jié)果一致[49-50]。

4 阻抗技術(shù)區(qū)分正常細(xì)胞與癌細(xì)胞

目前，臨床醫(yī)學(xué)領(lǐng)域診斷癌癥的方法包括超聲、放射和活檢等，活檢是確定癌變階段和程度的最精確的方法，然而有時也有假陰性判斷，而且需要諸如免疫組織化學(xué) （IH）、顯色原位雜交（CISH）或熒光原位雜交（FISH）等程序[63]。近年來，微管吸吮（MA）、蠕變壓縮、原子力顯微鏡（AFM）、光鑷技術(shù)等也被用來評估細(xì)胞的癌變程度[64]。然而，比較經(jīng)濟(jì)、非侵害、更加快速的診斷方法，是應(yīng)用阻抗技術(shù)來探知細(xì)胞的相關(guān)信息[65-71]。Han等將四種細(xì)胞：正常乳腺細(xì)胞（MCF-10A）、早期乳腺癌細(xì)胞（MCF-7）、侵入性人類乳腺癌細(xì)胞株（MDA-MB-231）和轉(zhuǎn)移性人類乳腺癌細(xì)胞株 （MDA-MB-435），捕獲到空腔

內(nèi)，并測定它們的阻抗，結(jié)果表明，與正常乳腺細(xì)胞相比，癌細(xì)胞的電容值分別下降4.1%，16.0%和19.1%[65]。Kang等在此基礎(chǔ)上，提高了檢測裝置的穩(wěn)定性和靈敏度，測定出正常乳腺細(xì)胞（MCF-10A）和早期乳腺癌細(xì)胞（MCF-7）阻抗實部和相位角的平均差異分別是 44.4 Ω和1.41°[66]，如圖6。之后，這個課題組又利用相同的裝置區(qū)分了正常前列腺細(xì)胞（RWPE-1）和其癌細(xì)胞（PC-3），兩者的導(dǎo)納和電納分別相差54.55%和54.59%，重現(xiàn)性優(yōu)異[67]。Chandra等報道了基于功能化的磁性納米顆粒制成的生物阻抗傳感器，進(jìn)行宮頸癌的早期檢測。宮頸癌細(xì)胞可以有選擇性地通過修飾電極檢測到，這在癌癥的監(jiān)測和臨床治療方面有巨大的發(fā)展前景[68]。利用阻抗技術(shù)區(qū)分正常細(xì)胞和癌細(xì)胞已受到廣泛關(guān)注，如何進(jìn)一步提高準(zhǔn)確度和靈敏度，為癌癥早期檢測提供預(yù)警信息，是目前的研究重點。

圖6 乳腺癌細(xì)胞MCF-10A和正常乳腺細(xì)胞MCF-7阻抗值和相位角隨頻率變化的差異：(a)阻抗實部；(b)阻抗虛部；(c)阻抗模值；(d)相位角[66]Fig.6 Electrical impedance responses of MCF-10A and MCF-7 as a function of frequency:(a)real part(b) imaginary part;(c)magnitude and(d)phase angle.The vertical bars represent the error defined by maximum and minimum values.The insets show the precise values when the target cells are distinguished at each signal

5 展望

目前，細(xì)胞阻抗傳感技術(shù)已在細(xì)胞測定方面展示出了巨大前景，其無需標(biāo)記、非破壞性、操作簡便等特點使得其廣泛應(yīng)用于臨床和科研領(lǐng)域，包括細(xì)胞狀態(tài)和行為的監(jiān)測、藥物測試、正常細(xì)胞與癌細(xì)胞的區(qū)分等。然而，目前的檢測體系大多是將很多細(xì)胞培養(yǎng)在一個電極上，因此阻抗響應(yīng)反映的是細(xì)胞的整體狀態(tài)，而無法提供單細(xì)胞信息，如何發(fā)展超靈敏電極傳感平臺以實現(xiàn)單細(xì)胞的檢測是今后研究的重點。

隨著檢測技術(shù)的快速發(fā)展，細(xì)胞阻抗傳感裝置也逐漸商業(yè)化，性能更加穩(wěn)定，操作更加方便，然而可以進(jìn)一步微型化、自動化，設(shè)計成各種功能化芯片，供應(yīng)各種檢測需要，降低成本，減少檢測時間，實現(xiàn)高通量檢測。

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The application process of impedance sensing in cell detection

Hu Qing-qing,Cui Yao,Zhang Fan*,He Pin-gang*
(School of Chemistry and Molecular Engineering,East China Normal University,Shanghai 200241,China)

Electric Cell-substrate Impedance Sensing is the combination of cell culture and impedance technology. Relevant cells cultured on working electrode are subjected to an alternating electric field,when cells attach and spread on this electrode,current signals of the whole system are collected to perceive cells state.With advantages like real-time,continuous,non-invasive,and label-free,impedance technology has been widely used in cell properties determination,cancer cells detection and anticancer drug screening etc.In this paper,we briefly review the recent application process of impedance in cell detection,involving the design of the detection system,the impedance monitoring of the physiological state of cells,the application of impedance in cytotoxicity and drug test, the differentiation of normal cells and cancer cells by impedance.

impedance;cell detection;review

國家自然科學(xué)基金資助項目（21405049）；上海市教育委員會科研創(chuàng)新項目（13zz032）

*通信聯(lián)系人，E-mail:fzhang@chem.ecnu.edu.cn;pghe@chem.ecnu.edu.cn