Robert Blick 11/3/06Rechenelemente für das 21. Jahrhundert: Vonder Einzel-Elektronen-Schaltung zum Bio-Transistor 1 Madison /06On-chip recordings of single ionchannelsRobert H. Blick, Nakul Shaji, Hyun-Seok Kim, Minrui Yu, Hua Qin.Electrical and Computer EngineeringUniversity of Wisconsin-MadisonIn collaboration with:Dan van der Weide, UW-ECENiels Fertig, Nanion Technologies GmbH, GermanyGeorgyi Los, Promega Inc., Madison, WI.work supported by DARPA (MOLDICE) and NSF (MRSEC, IRG1)earlier by DFG and BMBF, Germany. Madison /06Experimente an Zellen: Das Ziel ist der SealFrom Sakmann & Neher, Scientific Americanpatch pipette attached tocell membrane with embedded ion channels1. why ion channels... Madison /06classical patch clamping on cells: xzyObj. Madison /06patch clamping: resolution and limitationsco1o220 ms2 pA Madison /06xzyObj.2 µm2 µmChip statt Pipettepatch clamping: new & oldBlick / Fertig / Behrens (1998) Madison /06advantages of the patch clamp chipsim-noise• low cross capacitance < 1 pF• lower series resistance ~ 3 MΩ• lower noise level ~ 300 fA (fc= 3kHz)• planar geometry• accessible from bothsides• applicable for HTS• applicable for high-frequency recordingsRobert Blick 11/3/06Rechenelemente für das 21. Jahrhundert: Vonder Einzel-Elektronen-Schaltung zum Bio-Transistor 2 Madison /062. preparation techniques and substrates applied:siliconquartzborosilicatelithographyfocused ion beamion track etchingetc. Madison /062000 µmmicrostructured patch clamp probes:2 µmpossible substrates:-Si/SiO2/Si3N4+ : standard MEMS- : high density of charge carriers,- : little surface for seal formation- : expensive material-glass (borosilicate / quartz)+ : excellent insulation, low cap.+ : proven to seal membranes+ : inexpensive material- : non-standard processing 200 µmCelllow capacitancelow access resistancelow noise Madison /0620 µm1 µmSiSi3N4Si3N4vertical section450 µmpatch clamp fabricated in silicon-substrate• low n-doped Si-substrate• Si3N4 deposition by LPCVD• anistropical V-groove etch• etching of Si3N4-membrane Madison /06integration of chip into setup10 µmpipetteC6-glioma cellsuspendedSi3N4 layerchip is mounted in a custom made holdermanipulation of a cell on the chip via a suction pipette Madison /06aperturerim ofsuspendedareadebrisFertig et al., Appl. Phys. Lett. 77, 1218 (2000)first membrane patch on Si-substrate20 µm1 µm Madison /06patch clamp chip fabricated from crystalline quartzthinningRobert Blick 11/3/06Rechenelemente für das 21. Jahrhundert: Vonder Einzel-Elektronen-Schaltung zum Bio-Transistor 3 Madison /06500 nm20 µmapertures in crystalline quartz substrate (two side etch) Madison /06chip structuring by Focused Ion Beam (FIB) FIB allows definiton of fine apertures with sub-micron resolution,disadvantage is the low rate of material removal.2 µm2 µm2 µm1 µm Madison /06Latent ion track etching applied for glass structuringLokale Störungen im Substratnach Passage eines schweren,hochenergetischen Ions. Der gestörte Bereich (die latente Spur)lässt sich selektiv ätzen, so dass je nach Ätzparametern eine zylindrische, bzw. konische Pore entsteht.highly accelerated ionglass is then much more susceptible to HF etch Madison /06cylindrical pores in polycarbonate (NaOH-etch) Madison /06200 µm20 µm10 µm1 µmSingle ion tracketching:Au+18Fertig et al., Phys. E Rap. Comm. 64, 040901 (2001). micro apertures in glass substrates Madison /063. experiments:on artificial lipid membranes / on cells /and high-frequency measurementsRobert Blick 11/3/06Rechenelemente für das 21. Jahrhundert: Vonder Einzel-Elektronen-Schaltung zum Bio-Transistor 4 Madison /06lipid membranes on microstructured chipscurrenttime Madison /06gramicidin dimersCs+-curents through gramicidine A in bilayer From DiPhPC. bilayer is painted on glass chip aperture with 2 µm diameter. The measurement is performed inCsCl-solution (1M) at 200 mV.above: filter 3 kHz,below: filter 1 kHz.noise level (rms) at 3 kHz: 320 fAwith bandpass (0.3-3 kHz): 260 fA. Madison /06measurements on gramicidinStröme durchGramicidin A.histogram on conductance Madison /06alamethicin as a gated model pore Madison /06conducting alamethicin forms channels# of alamethicinmonomere Madison /06measurements on alamethicin50 ms50 pACurrent @ 10 KHzalamethicin currents in 1M NaCl solutionRobert Blick 11/3/06Rechenelemente für das 21. Jahrhundert: Vonder Einzel-Elektronen-Schaltung zum Bio-Transistor 5 Madison /06experiments on cellsZellsuspension auf dem Chip.mount for chip with integrated electrodesand suction system.check www.nanion.de Madison /06cells in the aperture10 µm Madison /06voltage gated K-channels in CHO-cells400 pA80 mV20 msCHO-BK,IK(Ca) Madison /06high-throughput drug screening16 channels, 96-well compatiblerapid solution exchangelow volume consumptionsmall footprint, benchtop designmodular, scalable --> 384 Nan]i[on Technologies GmbH Madison /064. now: some new stuff...1. colloidal quantum dots on membranes as ICs2. semiconductor membranes in solution as sensors Madison /061. combining bio-membranes with inorganics quantumdots: tunable by electromagnetic fields, fluorescencereadout – dots form ion channels 50 nm A 50 nm A B (i)(ii)(iii)100 (m s)10 (pA) 50 nm A B (i)(ii)(iii)100 (m s)10 (pA) 50 nm A B (i)(ii)(iii)100 (m s)10 (pA) 50 nm A B (i)(ii)(iii)100 (m s)10 (pA) 50 nm A B (i)(ii)(iii)100 (m s)10 (pA) 50 nm A B (i)(ii)(iii)100 (m s)10 (pA)BBBBBBBRamachandran, Kumar, Blick, van der Weide, APL 86, 083901 (2005)Robert Blick 11/3/06Rechenelemente für das 21. Jahrhundert: Vonder Einzel-Elektronen-Schaltung zum Bio-Transistor 6 Madison /06 (i)(ii)(iii)100 (m s)10 (pA )1. combining bio-membraneswithinorganics quantum dots:0 -5 -10 -15 -20 -25 -30 -35 -4002004006008002000400060008000Number of eventsCurrent (pA)- 30 mV0 -5 -10 -15 -20 -25 -30 -35 -4002004006008002000400060008000- 40 mV0 -5 -10 -15 -20 -25 -30 -35 -4002004006008002000400060008000Number of eventsNumber of events- 50 mVohmic Madison /062. … outlook – membranes & topology Madison /062. Fabrication of elastic-strain-sharing SiNMsSiBuried oxideSiBuried oxideSi49% HF35 nm Si40 nm Si150 nm Si0.84Ge0.1635 nm Si40 nm Si150 nm Si0.84Ge0.16New host(A) Deposit Si/SiGe/Si by CVDor MBE on SOI substrate.(B) Pattern access holes toburied oxide.(C) Remove buried oxide withHF.(D) Transfer membrane to newhost substrate. Can bemany different materials.Membrane thicknesses: 200nm 10s of nmAlso
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