Oscillating high-aspect-ratio monolithic silicon nanoneedle array enables efficient delivery of functional bio-macromolecules into living cells

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1 Oscillting high-spect-rtio monolithic silicon nnoneedle rry enles efficient delivery of functionl io-mcromolecules into living cells Disuke Mtsumoto,, Rmchndr Ro Sthuluri, Yoshio Kto, Yron R. Silererg, Ryuzo Kwmur, Futoshi Iwt, Tkeshi Koyshi nd Chikshi Nkmur, Deprtment of Biotechnology nd Life Science, Tokyo University of Agriculture nd Technology, -- Nk-cho, Kognei, Tokyo, 8-888, Jpn Biomedicl Reserch Institute, Ntionl Institute of Advnced Industril Science nd Technology (AIST), Centrl -- Higshi, Tsuku, Irki, -8, Jpn Deprtment of Mechnicl Engineering, Shizuok University, -- Johoku, Hmmtsu -8, Jpn Reserch Center for Uiquitous MEMS nd Micro Engineering, AIST, -- Nmiki, Tsuku, Irki -8, Jpn

2 Supplementry Figures Supplementry Fig. ) FE-SEM imges of nnoneedles. A constnt etching time often resulted in nnoneedles with overly-nrrow ottom prt (left). Grdully decresing the etching time resulted in more uniform nnoneedle (right). ) FE-SEM imges of fricted micropillr nd nnoneedle rrys. Micropillr rry (top) nd nnoneedle rry (ottom) re spced out uniformly. Scle rs in nnoneedle- nd micropillr-rry imges re µm, nd those in the single nnoneedle nd micropillr imges re µm. These smples re tilted.

3 Reltive Fluorescence Intensity (.u.) Reltive fluorescence intensity (.u.) μm pitch µm pitch Time (min) Supplementry Fig. In vitro ssy of moleculr econs immoilized on the nnoneedles surfce. ) XZ imges of µm pitch moleculr econ-immoilized nnoneedle rry, reconstructed from stck of CLSM imges efore (left) nd min fter (right) ddition of trget single strnd DNA ( -AACTTTGGTATCTTTGGTATCGTGGAAGGACTCATGACG). ) Time course of the reltive fluorescent intensities of the nnoneedles fter ddition of µm ssdna. The reltive fluorescence prior to trget sequence ddition is set s. Scle rs in re µm. 8 7 Inserted Not inserted Time ( min ) Supplementry Fig. Response of moleculr econ in vivo using µm pitch nnoneedle rry. ) YZ CLSM imges of MB-modified nnoneedle inserted into cytosol of HEK9 cell. Top imge ws scnned right fter insertion, nd ottom imge ws scnned min fter insertion. Yellow circle shows the outline of the cell. ) Time courses of reltive fluorescence intensities of the nnoneedles in the cytosol following insertion. Time-point indictes prior of nnoneedle insertion. Reltive fluorescence intensities re determined reltively to time-point. Scle rs in re µm.

4 Amplitude (.u.) Reltive fluorescence intensity 8 7 Time (min) cytosol nucleus no insertion Supplementry Fig. Lck of fluorescence response for the humn-specific moleculr econ when inserted into mouse NIHT cells. ) YZ CLSM imges of MB-modified nnoneedle inserted into cytosol nd nucleus of NIHT cell min (top) nd min (ottom) fter insertion. ) Time courses of reltive fluorescence intensities of the nnoneedles in the cytosol, nucleus, nd medium following insertion. Time-point indictes prior for nnoneedle insertion. Reltive fluorescence intensities re determined reltively to time-point. n= for ech condition. Error rs re SD. Scle rs in re µm Frequency (Hz) Supplementry Fig. Oscilltion resonnce frequency spectrum of the rry holder of the mnipultor. The hrmonic frequencies re detected t out 7 Hz, Hz, 7 Hz, Hz.

5 Supplementry Tles Supplementry tle Averged dimeters nd lengths of micropillr rrys in chips µm pitch micropillr rry µm pitch micropillr rry Chip Chip Chip Chip Chip Chip Chip Chip Chip Chip Dimeter (µm) SD CV (%) Length (µm) SD CV (%) Supplementry tle Averged dimeters nd lengths of nnoneedle rrys in chips µm pitch nnoneedle rry µm pitch nnoneedle rry Chip Chip Chip Chip Chip Chip Chip Chip Chip Chip Dimeter (nm) SD CV (%) Length (µm) SD CV (%)