TheBiophysical Society of Japan General IncorporatedAssociation Slcl-1 Femtoliter chamber for single-molecule analysis OHiroyuklNoji ISIR, Osaka Univ. Slcl-2 DigitalMicrotluidics OChang-JinKirn DepartmentofMcchanicalandAerospaceEngineering,UCLA Microlnano technotogy enables te fabricate miero/nanometer-sized devices fur fast and highly scnsitjvc dctection ot' biological reaction. Wit: dcvclupcd micronsized reaetien chambers to cnclese reactien mixture with a volumc ef a few femtoliter. Such an

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... xtremely small reaction chamber allows us to detect very small amount of chemical producls generated by u singie enzyme molecL]ie, The enzyrnatic reaction activities of beta-galactosidase and horse radish peroxidase were detectecl at a single molecule level by entrapping fiuorogenic reaction ntixture. Combinution ef this highty seusitive detectien methed with a single-meEecule manipulatiom uf a rotur>・ mDleculur motor, Fl-ATPuse effers the vv'ay to the single-moleculeanatysisefchemomechanicaicouplingefficiencyofF1-MI'ase.Recenrty, another grovp reported that stochastic expression of protein iriolec-les in a single bacteria celt was monitored at a single-motecule level using similar reaction chumber These expsrime]ts certify the lar.oe potential of the chumber for highly se"sitiveanal)・sist)fbiel{)gicalreactiens. Digital microfluidics is aii emerging class of microfiuidics technoleg)z where fluidg arc hand]ed in discrete volumcs rather than eontinunus flows. For pr[messing of liguids, dToplats can be moved by various actuatlon meLhods, 1iicluding thermal, surface wave, e]ectrostatic, dietectrophoTetic, and electrowettiiig, currently lhe tast being the most ec)mm{m. A comrnon feutuTe c]f the dreplet-driving sehernes is that the actuations occur locally at each droplet. Since droptets are not meved by the pressure around them. digita1 microfiuidic systems ean be built witheut microchannels, pressure sources (e.g., micropumps) or regulalory etements (e..g,. microvalve,s), greatl], simplifying the devices and systems. Our current emphasis on digital microfiuidics is to establish a lab-en-a-chip platforni by the mechanism of electtowetting-on-dielectric (EWOD), Demonstrated to nianipulate aqveous droplets in the air or ln oil, liWOD-based microfiuidics development has accomplished man}, physical funetieiialities -ereating. dividing, and merging droplets, tnixing different droplets, separating and concentrating partieles in a dreplet, and printing such drepfets. As a biochernical applicatien example of the EWOD chip, we clemonstrate on-chip sample processing for MALDI Mass SpectroTnetry. To demonstrate the simplicity EWOD digiud microfiuidics allows fer H},stem devetopment, we sho-'case a stand-u]one handhetd proLotype system complete with a battery' pack. With the abitity to cTeate EWOD chips capable of manipu]ating multiple droplets on a t-・o-dimensionat gTid array, build an entire systetn en a printed circuit beard. and accurateay cemrol droptet velullies on chip threugh real・・time feedback, a eumplele handbeld lab-on-a-chip system seems withinthehorizell. Circuits (BioASICs) and quantt]m nanoplasmonic prebes such as nanestn]ctured suiface enhanced Raman seatterimg (SERS) substrates are deve]oped. S(ijlr.s'pate HioASICs aTe created by connecting existing znd navet micTofluidic circuitg for high-content experimental bieleg}' in new ",ays. We ure creating a library of these "building btocks'i to devetep multifunctional biologicat mieroprocessors, To build a sotid foundation of futuTe high-speed inicro-and nanofluidic bioprocessors for experiniental systems biology and biomarker discovery. we have deyeloped design rules and critical modules ot' BioASICs such as single cell analysis chip, integrated multiple patch-clamp aJTay, dynarmic cell culture array, on-cbip cell lysing device, sample preparation chip. cell separatlon device, high-density single cell analysis chip,mc)lecularharvestingdevice,cel1-cel1commu"icationaTmay.1<ecemtly,weals" accemptished artificial lis・ers en a chip fo]' drug screening und drug discovery. FoT llallusuale speutroscopic moleuLLIar imaging und phutothermul therapeutic applications, nanocrescent SERS probes are developed, The formation of asyminetric nanophotonic crescent structure is accomplished by the interfacgng both bottom-up and top-down metliods, which allews te ereate effective local field eiihamcement sriuctures, batch ianofabrication, and precise controls ot' hot, spot coupling distaJ]ce for in-vivo mulecular imaghig. Gold-based llaiiocrescents have structures with a sub-10 nm sharp edge, which can enhance lecal etectromagnetic field at the edge area. ']'he advanced nanocresceiit SERS prebes ca" be upplied foT scnsitive molccular dctection arid electron transfbrs ef biemoleculos. In additien, intcgratcd nanotluidic SERS devicc can proyide a ncw sulutio] for label-i'ree genoinics and proteomics, The funcrieiial BieASICs and quantum nanoplasmonics have a petential to impact en systems bie]ogy, quantitative cell biology, biophysics. andmoleclllarmedicine. Memhrane proteins ptay very irnportanl roles in cells (e.g. recognitien or transportation of motecuies). They are also useful in varieus industrial fields, including iiext-gcneratiun diagnosis Lcchnigucs. drug discovcry, and highly senshis'e ien-channel-based bioseiisers. in this presentation, I wtll ;lltroduce our approach toward rnembrane protein chips: an urray of singte-species-specific membrane proteins reconstituted into plunar lipid bilayers formed in microfabricated holes and channe]s. In this approaeh, a highLy rcproducibLc rncthod was developed for p!anar lipid bilayer reconstinition. Planar tipid bilayers are d'ormed at aperLures. 1OO micren in diarnetei', by fiowing lipid organic solution and buffer ulternulely into un integrated rnjcrofiuidje chunnel, Using this technique, multiple lipid bilayers are fermcd simultancously in a singlc cbip, and channcl currents through pcptide ien charmcls was recordcd to prove the cempatibility of the chip with single molecule electrophysiology We belieye thut these devices ure usefu1 for an efflcient and rapid analysis of single-species-specific membrane proteins,