ETTERS NATURE NANOTECHNOLOGY DOI: 10.1038/NNANO 2011.217 100: 1 ratio as for the results presented in Fig. 4 for 1 M/10 mM) Methods show that the signal amplitude is approximately constant when The silicon nanowires were synthesized by chemical vapour deposition(CVD) the buffer concentration changes proportionally in both chambers, methods as described previously".Electron-beam lithography(EBL)and solid-state conventional charge-based FET sensing mechanism. Furthermore, deposited by plama-enshanced cvp was ned to pais ate al metal meatrbodes when the cis/trans concentrat ratio is reduced to 10:1. the bef recorded translocation signal (Supplementary Fig S7)is reduced a JEOL 2010F field-emission TEM. The nanowire- s predicted by the model in Fig. 3c. Finally, translocation experiments carried using the formally neutral polymer polyethy lene glycol ( Supplementary Methods, Fig. S8)show correlated clamped-on PDMS chambers with a tight seal, which were filled with sterilized and filtered buffer solutions. DNA translocation measurements were made using ionic current and FET conductance signals similar to but linearized pUC19 FET and ionic current signals were fied and digitized using smaller in amplitude than recorded for DNA translocation. The standard electronics with the sen smaller FET signal is consistent with expectations for our model See supplementary information for full methods and any associated references. (given the smaller ionic current signal change)and,more importantly, the fact that we observe the same signal polarity in Receive September 2011; accepted 7 November 2011 the FET channel is inconsistent with a charge-based FET sensing publish ine 11 December 2011 mechanism but in complete agreement with our potential sensing mechanism References A key advantage of the nanowire-nanopore FET sensor is 1. Branton, D et al. The potential and challenges of nanopore sequencing. Nature biotech.26,1146-1153(2008) sis chamber without complex microfluidic systemas. Notably, 2. Nantkrt an B. .& 615-624 anopore sensors for nucleic acid analysis. simultaneous recording from three nanowire-nanopore devices 3. Zwolak, M.& Ventra, M D Colloquium: physical approaches to DNA observed in all three FET channels as well as for the total ionic 4. Dekker. ic ond detection. Rev. Mod. Phys 80, 141-163(2008) current channel. Closer examination of the three fet and total 5. Clarke, J et al. Continuous base identification for single-molecule nanopore DNA sequencing. Nature Nanotech. 4, 265-270 (2009). nic current signals (Fig. 4b)shows clearly that the three FET 6. Derrington, L M et al. Nanopore DNA sequencing with MspA.Proc. NatlAcad. channels operate independently, and every falling or rising edge ScL USA107,16060-16065(2010) apparent in the total ionic current channel can be uniquely corre- 7. Garaj, S. et al. Graphene as a subnanometre trans-electrode membrane Nature lated to a corresponding edge in one of the three FET channels 467,190-19302010) Significantly, the total ionic current signal reconstructed from the 8. Merchant, C. A. et al. DNA translocation through graphene nanopores, data for the three FEt channels(dashed-red trace, top panel, 9. Schneider, G E et al. DNA translocation through graphene nanopores. ig, 4b)exhibits nearly perfect agreement with the measured total Nano Lett10,3163-3167(2010) ionic current(Supplementary Methods). In addition, a histogram 10. Fologea, D, Uplinger, J, Thomas, B, McNabb, D.S.& Li, J. Slowing D channel-specific ionic currents (Supplementary Methods, translocation in a solid-state nanopore. Nano Lett. 5, 1734-1737(2005). ig. S9)demonstrates that the ionic current signa litudes in 11. Peng, H& Ling X S Reverse DNA translocation throu solid-state nanopore different channels are also independent. As previous studies have 12. Olasagasti, E. et al. Replication of individual DNA molecules under electronic feT devices2with reproducible properties, and the local potential 13. Luan, B et al. Base-by-base ratcheting of single stranded DNA through a gnal decay length is as short as tens of nanometres, we expect nat it will also be possible to multiplex the nanowire-nanopore through a semiconductor nanopore-capacitor. Nanotedhnology 17, 622-633(2006). th FETs in much higher numbers and densities. Direct sequencing of long single-stranded DNA molecules using ys.Rev.Let95,2l6103(2005 FET-based nanopore sensors and the new potential change 16 Lagerqvist, J, Zwolak, M. Ventra, M D Fast DNA sequencing via transverse detection mechanism will require optimization of the signal-to- electronic transport. Nano Lett. 6, 779-782(2006) noise ratio associated with individual bases as well as improvement 11,279-285(2011) 17. Ivanov, A.P. et al. DNA tunnelling detector embedded in a nanopore Nano Left. in signal spatial resolution. Recognizing that direct base differen- 18. Chaste, I et al. Single carbon nanotube transistor at GHz frequency Nano Lett tiation by FET potential measurement is coupled to variations 8,525-528(2008). in the ionic current suggests that concepts proposed and demon- 19. Hu, Y, Xiang. J Liang, G, Yan, H. Lieber, C M Sub-100 nanometer channel strated for base-resolved ionic current measurements5-7 could successfully combined with our work. For example, it should be 20. Cui, Y. Wei, Q, Park, H. Lieber. C. M Nanowire nanosensors for highly possible to extend our nanowire-nanopore FET to atomically thin sensitive and selective detection of biological and chemical species. Science 293 graphene membranes- so as to achiev 1289-1292(2001) resolution, although the graphene nanopore would require precise 21 Patolsky, F et al Electrical detection of single viruses. Proc.Nat! Acad. Sci. USA structure engineering to enable differentiation of the distinct 101,14017-14022(2004) 22. Patolsky, F. et al Detection, stimulation, and inhibition of neuronal signals bases.Alternatively, coupling an engineered protein nanopore to with high-density nanowire transistor Science313,1100-1104(2006 the nanowire-nanopore FET could provide both the spatial and 23. Kim, M I, Wanunu, M. Bell, D. C. Meller, A Rapid fabrication of uniformly base resolution necessary for direct sequencing due to the localized sized nanopores and nanopore arrays for parallel DNA analysis. Adv. Mater. nanowire-nanopore FET sensor results and modelling strongly 24. Kasianowicz, - J Brandin, E, Branton, D &Deamer, D. WCharacterization of ad.Sa.UsA93,13770-13773(1996) urrent and other sensor-based detection schemes, including 25. Li, J et al. Ion-beam sculpting at nanometre length scales. Nature 412, rger measurement signals, high signal bandwidth with attractive 166-169(2001). nanopore-size scaling, and straightforward integration and multi- 26.Cohen-Karni,T,Timko,BP,Weiss,LE&Lieber,CMFlexible electrical plexing. We believe that this work provides a strong starting point reording from cells using nanowire transistor arrays. Proc Natl Acad. Sc: USA for a new class of nanopore sequencing devices with the capability 27. Tian, B et al. Three-dimensional, flexible nanoscale field-effect transistors as for fast direct sequencing and large-scale integration localised bioprobes. Science 329, 831-834(2010) NatureNanotEchnOlogYIVol7IFebRuaRy2012Iwww.nature.com/naturenanotechnology o 2012 Macmillan Publishers Limited. All rights reserved100:1 ratio as for the results presented in Fig. 4 for 1 M/10 mM) show that the signal amplitude is approximately constant when the buffer concentration changes proportionally in both chambers, in agreement with the model predictions and in contrast to the conventional charge-based FET sensing mechanism. Furthermore, when the cis/trans concentration ratio is reduced to 10:1, the recorded translocation signal (Supplementary Fig. S7) is reduced as predicted by the model in Fig. 3c. Finally, translocation experiments carried using the formally neutral polymer polyethy￾lene glycol (Supplementary Methods, Fig. S8) show correlated ionic current and FET conductance signals similar to but smaller in amplitude than recorded for DNA translocation. The smaller FET signal is consistent with expectations for our model (given the smaller ionic current signal change) and, more importantly, the fact that we observe the same signal polarity in the FET channel is inconsistent with a charge-based FET sensing mechanism but in complete agreement with our potential sensing mechanism. A key advantage of the nanowire–nanopore FET sensor is the potential for integration and multiplexing within a single analy￾sis chamber without complex microfluidic systems28. Notably, simultaneous recording from three nanowire–nanopore devices (Fig. 4a) demonstrates that continuous translocation events are observed in all three FET channels as well as for the total ionic current channel. Closer examination of the three FET and total ionic current signals (Fig. 4b) shows clearly that the three FET channels operate independently, and every falling or rising edge apparent in the total ionic current channel can be uniquely corre￾lated to a corresponding edge in one of the three FET channels. Significantly, the total ionic current signal reconstructed from the data for the three FET channels (dashed-red trace, top panel, Fig. 4b) exhibits nearly perfect agreement with the measured total ionic current (Supplementary Methods). In addition, a histogram of channel-specific ionic currents (Supplementary Methods, Fig. S9) demonstrates that the ionic current signal amplitudes in different channels are also independent. As previous studies have shown that it is possible to fabricate large numbers of nanowire– FET devices29 with reproducible properties, and the local potential signal decay length is as short as tens of nanometres, we expect that it will also be possible to multiplex the nanowire–nanopore FETs in much higher numbers and densities. Direct sequencing of long single-stranded DNA molecules using FET-based nanopore sensors and the new potential change detection mechanism will require optimization of the signal-to￾noise ratio associated with individual bases as well as improvement in signal spatial resolution. Recognizing that direct base differen￾tiation by FET potential measurement is coupled to variations in the ionic current suggests that concepts proposed and demon￾strated for base-resolved ionic current measurements5–7 could be successfully combined with our work. For example, it should be possible to extend our nanowire–nanopore FET to atomically thin graphene membranes7–9 so as to achieve single base spatial resolution, although the graphene nanopore would require precise structure engineering to enable differentiation of the distinct bases. Alternatively, coupling an engineered protein nanopore to the nanowire–nanopore FET could provide both the spatial and base resolution necessary for direct sequencing due to the localized change of the potential at the nanopore opening (Fig. 3d). Our nanowire–nanopore FET sensor results and modelling strongly motivate such effort, given the advantages over direct ionic current and other sensor-based detection schemes, including larger measurement signals, high signal bandwidth with attractive nanopore-size scaling, and straightforward integration and multi￾plexing. We believe that this work provides a strong starting point for a new class of nanopore sequencing devices with the capability for fast direct sequencing and large-scale integration. Methods The silicon nanowires were synthesized by chemical vapour deposition (CVD) methods as described previously30. Electron-beam lithography (EBL) and solid-state diffusion of the nickel contact were used to fabricate short-channel devices on commercially available SiNx TEM membrane grid chips. A SiNx conformal thin film deposited by plasma-enhanced CVD was used to passivate all metal electrodes before final lift-off. Nanopores were drilled by focusing the 200 keV electron beam in a JEOL 2010F field-emission TEM. The nanowire–nanopore FET sensor chip was glued onto a home-made PCB chip carrier and electrically connected to the chip carrier by wire bonding. The chip carrier was sandwiched between mechanically clamped-on PDMS chambers with a tight seal, which were filled with sterilized and filtered buffer solutions. DNA translocation measurements were made using linearized pUC19. FET and ionic current signals were amplified and digitized using standard electronics with the sensor set-up mounted in a Faraday box. See supplementary information for full methods and any associated references. Received 16 September 2011; accepted 7 November 2011; published online 11 December 2011 References 1. Branton, D. et al. The potential and challenges of nanopore sequencing. Nature Biotechnol. 26, 1146–1153 (2008). 2. Venkatesan, B. M. & Bashir R. Nanopore sensors for nucleic acid analysis. Nature Nanotech. 6, 615–624 (2011). 3. Zwolak, M. & Ventra, M. D. Colloquium: physical approaches to DNA sequencing and detection. Rev. Mod. Phys. 80, 141–163 (2008). 4. Dekker, C. Solid-state nanopores. Nature Nanotech. 2, 209–215 (2007). 5. Clarke, J. et al. Continuous base identification for single-molecule nanopore DNA sequencing. Nature Nanotech. 4, 265–270 (2009). 6. Derrington, I. M. et al. Nanopore DNA sequencing with MspA. Proc. Natl Acad. Sci. USA 107, 16060–16065 (2010). 7. Garaj, S. et al. Graphene as a subnanometre trans-electrode membrane. Nature 467, 190–193 (2010). 8. Merchant, C. A. et al. DNA translocation through graphene nanopores. Nano Lett. 10, 2915–2921 (2010). 9. Schneider, G. F. et al. DNA translocation through graphene nanopores. Nano Lett. 10, 3163–3167 (2010). 10. Fologea, D., Uplinger, J., Thomas, B., McNabb, D. S. & Li, J. Slowing DNA translocation in a solid-state nanopore. Nano Lett. 5, 1734–1737 (2005). 11. Peng, H. & Ling X. S. Reverse DNA translocation through a solid-state nanopore by magnetic tweezers. Nanotechnology 20, 185101 (2009). 12. Olasagasti, F. et al. Replication of individual DNA molecules under electronic control using a protein nanopore. Nature Nanotech. 5, 798–806 (2010). 13. Luan, B. et al. Base-by-base ratcheting of single stranded DNA through a solid-state nanopore. Phys. Rev. Lett. 104, 238103 (2010). 14. Gracheva, M. E. et al. Simulation of the electric response of DNA translocation through a semiconductor nanopore-capacitor. Nanotechnology 17, 622–633 (2006). 15. King, G. M. & Golovchenko, J. A. Probing nanotube–nanopore interactions. Phys. Rev. Lett. 95, 216103 (2005). 16. Lagerqvist, J., Zwolak, M. & Ventra, M. D. Fast DNA sequencing via transverse electronic transport. Nano Lett. 6, 779–782 (2006). 17. Ivanov, A. P. et al. DNA tunnelling detector embedded in a nanopore. Nano Lett. 11, 279–285 (2011). 18. Chaste, J. et al. Single carbon nanotube transistor at GHz frequency. Nano Lett. 8, 525–528 (2008). 19. Hu, Y., Xiang, J., Liang, G., Yan, H. & Lieber, C. M. Sub-100 nanometer channel length Ge/Si nanowire transistors with potential for 2 THz switching speed. Nano Lett. 8, 925–930 (2008). 20. Cui, Y., Wei, Q., Park, H. & Lieber, C. M. Nanowire nanosensors for highly sensitive and selective detection of biological and chemical species. Science 293, 1289–1292 (2001). 21. Patolsky, F. et al. Electrical detection of single viruses. Proc. Natl Acad. Sci. USA 101, 14017–14022 (2004). 22. Patolsky, F. et al. Detection, stimulation, and inhibition of neuronal signals with high-density nanowire transistor arrays. Science 313, 1100–1104 (2006). 23. Kim, M. J., Wanunu, M., Bell, D. C. & Meller, A. Rapid fabrication of uniformly sized nanopores and nanopore arrays for parallel DNA analysis. Adv. Mater. 18, 3149–3153 (2006). 24. Kasianowicz, J. J., Brandin, E., Branton, D. & Deamer, D. W. Characterization of individual polynucleotide molecules using a membrane channel. Proc. Natl Acad. Sci. USA 93, 13770–13773 (1996). 25. Li, J. et al. Ion-beam sculpting at nanometre length scales. Nature 412, 166–169 (2001). 26. Cohen-Karni, T., Timko, B. P., Weiss, L. E. & Lieber, C. M. Flexible electrical recording from cells using nanowire transistor arrays. Proc. Natl Acad. Sci. USA 106, 7309–7313 (2009). 27. Tian, B. et al. Three-dimensional, flexible nanoscale field-effect transistors as localised bioprobes. Science 329, 831–834 (2010). LETTERS NATURE NANOTECHNOLOGY DOI: 10.1038/NNANO.2011.217 124 NATURE NANOTECHNOLOGY | VOL 7 | FEBRUARY 2012 | www.nature.com/naturenanotechnology © 2012 Macmillan Publishers Limited. All rights reserved