TAl0 Proceedings of 2006 International Conferenceon Microtechnologies in Medicineand Biology

Okinawa, Japan 9-12 May 2D6

A SILICON-BASED SINGLE-CELL ELECTROPORATIONMICROCHIP FOR GENE TRANSFER

Younighak Choi, Brno Le pioufle2, Nbuyuki Takamai, and Beomjoon Kimi

'CIRMMJJS, The Univemsity of TokRyo4-6-1, Komaba, Meguro-ku, Toklyo Japan

Tel +81-3-5452-6224, Fax +81-3-5452-6225, E-mail bi6onkih itohy2LIMMS/CNRS-JJS (UVil 2820), France

Abstract

In our contribtlion, we presen:t the fibrication of electroporation microchip in detail. The practical experimenits of sinle-cell

electropotation with our fabicated microchip will be carTied out. Electropoation test efficiency and cell viability tests will beprovided. This device enfiables to reduce the size of samuples and thius the use of small amont of regenits. It may also permitto avoid cell separation (tra.nsfcted cells versus non transfcted cells) encountered when taditional bulk electroporation is

held.

Keyords: Gene trans Mcrochip, Single-ce/I electrporation

1 INTRODUCTION

Transfction of gene into cells is now a central technique inmolecular biology and an essential prerequisite for genetherpy. To improve traisfction efficiency is a caillengefor successful delivery of foreign DNA into cells n vito.There are some chemical and physical techniques tointroduce compounds of biological and medical interest(such as plasmid DNA, RNA, drugs) to the cell cytoplasm.Among those, e1ectroporaftio is a powerf and widespreadtebhnique fbr the itroduction of DNA into various types ofcells in vitro and in vivo With low toxicity [1,2]. It ses hihelectric fields to induce breakdown of the cell membranelipid bilayer and formation of ttrasiett or permanent poresin the mebrante. Recentiy, single-cell electoporation usingelectrolyte-filled capillaries and miicrofabricated chips hasbeen investigated with high ierest. Successfl sirgle-cellelectroporation requires isolation and electrical fieldfocusing on the target cell [3]. Microfbricated devicesrender these goals achievable. Indeed, MIEMS tec6lmhologymakes possible to appracoh close to single cells, inmdiviualproteins, and DNA, allowing manipulatiohs and analysiswhich were almost impossible previously [4].In this paer, we will shox the electroporation in singularcell level with microfabicated device xvith low voltagehighfrequency pulse. This will not give so much damage tocell tfironh the eec1troporation. Also, de to hightransfection efficiency, there will be no need to di§scrimimatetie tra.usfcted cells after electroportion.

untiet(a) 1 st tpe of device

icroelectrodearray

IPDMSseali

Outet(b) 2d type of complete device

Figure 1. Schemratic view of two types of devices

1-4244-0338-3/06/$20.00 02006 IEEE 195

2 FABRICATIONS

As shown in Fig. 1, the device is composed by am icrochannel leading to cantilever-ype microelectrodespair designed fbr focusing the electric field. Low amplitude.voltage is applied between electrodes set, as an isolatedsingle cell is captured between tiem. Therefore, littledamage to cells during the electrop oration process and highefficient gen:e transfer are expected.In this paper, two kinds of device with diferent processwere fabricated. The differ6ene between two devices is thefbrication process of microc6hannel. Onie has themicrochannel made by silicon of substrate SOI waftr, asshown im Fig. 2. The other has the microchannel made bySU-8 polymer as shown In Fig. 3 and Fig. 4.in here, we present the fabrication process of device withSU-8 mnicrochannel. First, after the SiN layer was depositedon the top side of the SOI wafer by LPCVD, the top-side ofSOI wafer was pattemed and etched fbr fabrication ofmicrocanitiever. The twin microcantilever has 12 pim thick,and is 65 pm long and 15 pin wide, respectively. The gapbetween twin microcantilevers is 10-20 Pm, which is therange of sizes as target cells. Secondly, Cr/Au metalelectrode was deposited on individual microcantilever array.Fig. 3 shows the SEM photos of pattemred microelectrodeson microcantilever arrays and reference electrodes. Afterdeposition of electrodes, microchannels are pattemed usingthe transparent photo-plastic polymer (SU-8). It allowssimple batch fabrication based on spin coating andsubsequent near-ultraviolet exposure an1d development steps.Fig. 4 showsVthe microscopy images of microcanrilever6electrode arrays and microchannel. The microchannel is 4mm long1 , 20 m deep and 1040 jm wide. After the

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Figuire 2. SEM photos of silicon microchamicro cantilever elect1rode arras in ttype device

rigure .3. OVIw11VLLJ bIooiImicUrtcaI

and microchannel in 2'A type device

rigure 4. 1array. andafarray)

backside of SOI wafir was etched, Si02 layer was removedfbr outlet fboration. Fially, 1 1-Mercaptoundecanoic acidwith hydrophilic property is seW-Iassembled on the surficeof Si substrate beneath the nicrochannel fbr smoothflowing of cell solution.The device with SU-8 microchannel has some advantagesover the device witi Si microchannel. It is easy to changethe size of microchannel through one mask change, notchange of substrate, and easier to pattern the electrode on aplane than on an inclined slope. Moreover, it is possible todo reversible flutidic sealing between SU-8 and PDMS andto integrate the reference electrode with the twin electrodearray, as shown im Fig. 1(b) anid Fig. 4.

3 RESULTS ANT) DISCUSSIONS

We successfully :fbricated the microchip for single-cellelectroporation. fhrough modification of fabricationprocess, reference electrode could be also integrated inmullti-airrs of microchannels. Refiirence electrode isintegrated to improve the transfeti6on efficiency ofchemical compounds like GFP (green fluorescent protein)into the device. Moreover, the fiabrcation process of Zn4type device was simpler than that of 1t type device, due tothe usage ofpolymer channel faibication.The main differences between our microfabricatedsingle-cell electroporation: chip and traditional builkelectroporation chip are as followsi) the low voltage is applied to the systemii) the inhomogeneous electiic fieldiii) the short distance between the electrodes and the cell

That is, our device can capture and locally electroporatesingle cells. Oing to sharp edged twin microelectrode withsmill gap, it is possible to apply low voltage which does notinduce irreversible po6re formaionand cell death. Also, the

isk of exposure of the ce11 to potential harmful effects from

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takimg place at high voltage in queous solution can bereduced.To optimize dmimensions of cantilever, gap and shapes to getbetter electric field, theoretical calculation was carried outby uising Femlab simulation tool, before experinents. Fig. 5shows the Femlab simulation results of electrc field aolundmicrocantilever electrodes. When 0.7 V voltages wereapplied between twin electrodes, the maximunm electricfield at sharp edges is aouit 3104 V/tn. It means that the1ocalized electroporatioon im single-cell level can beachieved by low applied voltage. Tibs low applied voltageis enough to electroporate single cell with high electric fieldacross the cell membamne. This is within the voltage range(0.25-1V) that dielectrnc breakdown of menibrne occurs

[5].Single-cell electroporation enable to investigate theintracellula blochemistry of a cell. With microchi basedon MEMS technology, electroporation can be used for highthroughput screening of gene and protenm expression and fordrug discovery for intracl ilar target.In future, we will present the experiment results afterelectroporation with ourt fabricated devlce with 1low voltagehigh-freqency puilse. Red flutorescent IP will be used forelectroporation test, and cell viability will also be testedwith Ttypan blue. Afier idding GEP to the cells, 10-100cells will be cuiltured 'i other chamber during 36 hours ormore after eletropotration.

(a)SSIe view

(b) Top viewFigure 5. SiInulation results of electric fieldmicro cantilever electrodes (@ FEMLAB)

ACKNOWLEDGEMENTS

The anthors would like to thank Prof H. Fjita and Prof Y.Sakal a uS, and the stff of the ISc1leanroom for theirassistance. This research was generously supported byJapa Society for the Promotion ofScience (JSPS)

REFERENCES

[1] D. Luo and W.M. Sazm an, Nture B1otechno ogy, Vo .18 (2000) 33-37[2] Y. Huang and B. Ruibinsky, Blomeedical Microdevices,Vol. 2:2 (1999) 145-150[3] K. Nolkrantz, C. Faree, K.J. luIti;g, P. Rylander and 0.Orwar, Anial. Chem., Vol. 74 (2002) 43004305[4] S. Camou, A. Tixier-Mita, H. Ftujita andT.TFujii Jai. J.Appl. Physics, VoL 43 (2004) 5697-5705[5] J. Olofsson, K. Nolkrantz, F. Ryttsen, B.A. Lanbie, SQGWeber, 0. Orwa Curt; Opin. In Biotech., Vol. 14 (2003)29 34

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