Letter pubs.acs.org/NanoLett
Outside Looking In: Nanotube Transistor Intracellular Sensors Ruixuan Gao,† Steffen Strehle,†,§ Bozhi Tian,† Tzahi Cohen-Karni,‡ Ping Xie,† Xiaojie Duan,† Quan Qing,† and Charles M. Lieber*,†,‡ †
Department of Chemistry and Chemical Biology and ‡School of Engineering and Applied Science, Harvard University, Cambridge, Massachusetts 02138, United States S Supporting Information *
ABSTRACT: Nanowire-based field-effect transistors, including devices with planar and three-dimensional configurations, are being actively explored as detectors for extra- and intracellular recording due to their small size and high sensitivities. Here we report the synthesis, fabrication, and characterization of a new needle-shaped nanoprobe based on an active silicon nanotube transistor, ANTT, that enables highresolution intracellular recording. In the ANTT probe, the source/drain contacts to the silicon nanotube are fabricated on one end, passivated from external solution, and then time-dependent changes in potential can be recorded from the opposite nanotube end via the solution filling the tube. Measurements of conductance versus water-gate potential in aqueous solution show that the ANTT probe is selectively gated by potential changes within the nanotube, thus demonstrating the basic operating principle of the ANTT device. Studies interfacing the ANTT probe with spontaneously beating cardiomyocytes yielded stable intracellular action potentials similar to those reported by other electrophysiological techniques. In addition, the straightforward fabrication of ANTT devices was exploited to prepare multiple ANTT structures at the end of single probes, which enabled multiplexed recording of intracellular action potentials from single cells and multiplexed arrays of single ANTT device probes. These studies open up unique opportunities for multisite recordings from individual cells through cellular networks. KEYWORDS: Core/shell nanowires, nanobioelectronics, nanoelectronic device, nanosensor, intracellular action potentials
stolen
nanowire field-effect transistors (NW FETs) with a transistor or diode at the tip, where the nonlinear NW structure enables ready insertion into cells,7,9 and (ii) branched intracellular nanotube FETs (BIT-FETs), where the transistor remains outside of the cell but senses the intracellular potential via the solution inside the passive nanotube bridge.8 Here we demonstrate a conceptually new and practically simple FET based probe that consists of a single semiconductor nanotube. The underlying principle of the active nanotube transistor, ANTT, intracellular probe (Figure 1A) involves the fabrication of source/drain (S/D) contacts to one end of a silicon (Si) or other semiconductor nanotube and electrical isolation of these S/D contacts from surrounding medium such that the solution filling the interior of the nanotube can gate the transistor and the variation of interior electrochemical potential is then recorded as a change in device conductance. Hence, if the free end of an ANTT probe is inserted into the interior of an electrogenic cell, the time-dependent changes associated with an action potential spike will give rise to time-varying conductance signal that maps the intracellular action potential. However, if a similarly configured solid Si (or other semiconductor) NW is inserted into the cell, no signal would be observed since it would not be possible to “gate” the transistor. We note that for p-type Si
A
n electronic device that can interface to the intracellular region of a live cell has several important constraints, including (i) small size, which can minimize invasiveness and potentially allow contact to subcellular structures, (ii) high sensitivity with decreasing size, and (iii) capability to multiplex at both single cell and cell network levels.1,2 Well-established electrophysiological techniques for cellular recording, such as patchclamp micropipets,3 metal microelectrodes,4 and intracellular glass microelectrodes,5,6 have advanced the understanding of electrogenic cells, although also have limitations in terms of (i) decreasing signal and signal-to-noise (S/N) with decreasing probe size in the submicrometer regime and (ii) capabilities for multiplexed measurements on single cells as well as larger-scale multiplexing for cell networks. Recently, researchers have been addressing these issues with the development of novel transistor-based probes7−9 and improved designs for micro/nanoelectrodes.10−12 The micro/nanoelectrodes exploit structures projecting from chip plane to enable invagination of cultured cell membranes and measurements of intracellular-like action potentials,10−12 although as passive recording electrodes they are limited in terms of their potential for miniaturization. On the other hand, transistor-based probes can be reduced in size to at least the 10 nm scale.1,13 However, active transistor requires two contacts for operation, and until recently this constraint has made intracellular recording devices difficult to achieve. Our group has recently described two solutions to this conundrum involving the synthesis of (i) kinked © 2012 American Chemical Society
Received: April 30, 2012 Published: May 14, 2012 3329
dx.doi.org/10.1021/nl301623p | Nano Lett. 2012, 12, 3329−3333
Nano Letters
Letter
stolen Figure 1. Principle and fabrication of the ANTT probe. (A) Schematic view of an ANTT probe inserted into a cell and recording an intracellular action potential (Vcell vs time, t) as a conductance (G) change in the active FET region between S/D contacts. Sensitivity to voltage changes from the external extracellular environment is effectively eliminated by SU-8 passivation of the nanotube region around the S/D contacts. The nanotube is shown as a halfcylinder for clarity. (B) Overview of the steps used for ANTT probe fabrication:14 (1) Transfer of germanium/silicon core/shell nanowires (Ge/Si NWs) to a SU-8 layer that was deposited and prebaked on a sacrificial layer (colored silver). (2) Registration of positions of Ge/Si NWs and definition of the bottom SU-8 layer. (3) Definition of S/D metal contacts followed by the top SU-8 passivation layer. Final etching of the sacrificial layer and Ge core yields the Si ANTT probe. (C) Schematic of the completed ANTT probe following release from the substrate. (D) SEM image of an ANTT probe. Scale bar, 10 μm. Inset, zoom of the probe tip from the dashed red box. Scale bar, 100 nm.
by solution access to the Si nanotube interior. To rule out the possibility that the Ge-etching process degrades the SU-8 passivation, we carried out control experiments on a Si/Si intrinsic-core/p-shell NW structure, where the shell is similar to the Ge/Si NWs used to make ANTT devices. Significantly, the Si/Si control device with SU-8 passivated S/D contacts showed similar sensitivity to the Ge/Si device and little or no sensitivity change following the same etching conditions used to remove Ge and yield the ANTT device. Taken together these results show that (1) the top/bottom SU-8 passivation effectively isolates the FET channel from external solution potential changes, and (2) the ANTT structure has good sensitivity to potential changes coupled through solution in the nanotube interior. In addition, we have explored the capability of the ANTT device to detect chemical changes in pH sensing experiments. Measurements of the G as a function of stepwise increasing pH showed a systematic increase with the increasing negative charge on the SiO2 surface24 of the p-type nanotube interior. These data plotted as change in potential versus solution pH (Figure 2B) yield a device pH sensitivity of up to 37 mV/pH, a value that is consistent with previous results reported for Si NW devices.7 Measurements made before and after closing the nanotube device end with SU-8 (inset, Figure 2B) confirm that this change is indeed due to pH detection from the solution inside the nanotube as no variation in G was observed after blocking solution exchange. These results show the potential of the ANTT probe to detect chemical and biochemical changes as previously demonstrated by NW FET sensors,7,24 although we note that necessity for diffusion of analytes within the nanotube will reduce the temporal resolution compared to external surface binding. We explored the possibility of recording intracellular action potential with the ANTT probes using spontaneously firing chicken cardiomyocytes. In these experiments, ANTT probes relieved from the surface (e.g., Figure 1D) were modified with a phospholipid layer,7−9,25 cardiomyocytes were cultured on flexible sheets of polydimethlylsiloxane (PDMS) as described previously,25,26 and then the PDMS/cell sheet was moved to
nanotubes, as used in our proof-of-concept studies, a positive change of intracellular potential yields a negative change in device conductance (Figure 1A), although the conductance can be quantitatively converted to potential using water-gate calibration measurements.1,7−9 The fabrication of ANTT probes was carried out in several steps briefly illustrated in Figure 1B.14 First, germanium/silicon core/shell nanowires (Ge/Si NWs) were synthesized by nanocluster catalyzed vapor−liquid−solid (VLS) growth of Ge NWs followed by p-type Si shell deposition as described previously.15−18 The Ge/Si NWs were dispersed from isopropyl alcohol or contact printed19−22 onto a prebaked SU-8 layer, which was initially deposited on a sacrificial nickel relief layer. After defining the lower SU-8 passivation/isolation layer by electron beam lithography (EBL), S/D metal contacts (Cr/Pd/ Cr, 1.5/75/50 nm) followed by an upper SU-8 passivation/ isolation layer were patterned by EBL. Etching the nickel sacrificial layer and Ge core of the Ge/Si NW yielded the p-type Si ANTT probe (Figure 1C). Scanning electron microscopy (SEM) images of ANTT probes (Figure 1D) confirm the basic fabrication strategy and show clearly the nanotube structure. Comparison of SEM images of ANTT device before and after the Ge core etching (Supporting Information Figure S1) shows clearly the open tip and a “bright-to-dark” change in image contrast along the length of the structure that is indicative of an open nanotube from the tip through to the S/D metal contacts. The “bend-up” angles, typically 40−60° and height, usually 15− 30 μm in our design, are controlled by changing the length of the stressed bimetallic S/D arms as described previously.7 We have investigated the electrical properties of the probe structures fabricated in this manner in aqueous solution before and after etching the Ge cores. Prior to Ge-etching, conductance (G) versus water-gate potential (Vwg) data show only a small change with a sensitivity of ∼0.10 μS/V (Figure 2A). Notably, after etching the Ge core to form the ANTT device the watergate data exhibits a 30× sensitivity increase to 3.0 μS/V.23 These results are consistent with the increased gate coupling afforded 3330
dx.doi.org/10.1021/nl301623p | Nano Lett. 2012, 12, 3329−3333
836
IEEE SENSORS JOURNAL, VOL. 7, NO. 5, MAY 2007
Current DNA detection methods based on sequencing by synthesis rely on optical readouts; however, a direct electrical detection method for this technique is not available. Application of the label-free electrical detection method, charge perturbation detection, applied to sequencing by synthesis, could be used for direct electrical detection of enzymatically catalyzed DNA synthesis by induced surface charge perturbation to detect any enzymatic DNA or RNA synthesis, as well as other biochemical reactions based on similar principles [11]. III. BIOELECTRONIC SENSORS: DESIGN AND PERFORMANCE DATA In general, single sensor systems can only provide partial information on the state of the environment, while multisensor systems combine related data from multiple similar and/or different sensors. The goal of using multisensor systems is to provide synergetic effects that enhance the quality and availability of information about the state of the measurement environment [12]. A number of sensing organs of different physical quantities and environmental conditions are discovered in living beings. On the other hand, output signals from these organs–nerve impulses– can be picked up by state-to-the-art techniques. Further signal processing will define the connection between sensed quantity and output data of such biological sensors. As a result, they could be applied in addition to the known artificial sensors for intelligent sensing. The known amperometric techniques of BSs involve the Renview bight realizing method [9], it requires extra stimulating of the isolated nerve fiber and the other method needs additional reagents and applied voltage. Creative integration of microchip technologies and nanostructures is feasible. Combined with electroporation, electrokinetics also enables inclusion of molecular complexes inside the cells. Alternatively, functionalized nanoposts can be used to impale cells and relay information from the cell interior to nanoelectronic circuits. The challenge for the future of designing a nano-interface in a microfluidic chip to probe a living cell lies in seamlessly integrating techniques into a robust and versatile, yet reliable, platform [13]. The method of combining the bioelectric nature of NIs and synaptic currents between neighboring neurons with body-temperature PC and zero resistance input of the SuFET device in order to obtain most advantageous biosensor/transducer was recently advanced [6]. Transducing the vortical magnetic field from the ECs (BSs) by the PC that is wrapped around the nerve fiber or DNA sequence is executed similarly, but the PC is in nano dimension [14]. By using the said superconducting magnetometer with a room- temperature PC (SIM) [4], [6], it is possible to create the implantable transducer. On the other hand, neuroelectronic systems for two-way interfacing of the neuronal and the electronic components by capacitive contacts and by FETs with an open gate was developed [15]. They observed a wide spectrum of transistor signals monitoring the activity of neurons from leeches, snails, and rats. In order for the electronic component to function under cryogenic (superconducting) conditions, there are a number of methods to connect them in vivo through three types of contacts: biocompatible, flexible, and with increased adhesion [16]–[18].
Fig. 1. An organic SuFET device and its electrodes.
IV. THE AVAILABLE FET’S VARIANTS APPLICABLE AS SUFET Application of the SuFET’s modifications such as CMOSuFET (low Tc) [19] and coplanar SuFET (high Tc) [20] broadens the range of requirements which are being satisfied by the SuFETTr. Low-dimensional semiconductor nanostructures and organic molecules, which offer unique possibilities such as extremely low-power dissipation, quantum effects, surface sensitivity, and low synthesis cost, could be the building blocks for next-generation electronics [21]. Moreover, an organic superconductivity of carbon molecules, known as bucky balls, which can act as superconductors at relatively warm temperatures, raises hopes for loss-free organic electronics and their practical applications in biosensors, including organic ones. High densities of electrons and holes have been induced by gate doping in a FET geometry (Fig. 1). At low temperatures, the material turns superconducting with a maximum transition temperature of 117 K in hole-doped C /CHBr [22]. The increased spacing between the C molecules increases the denis well docusity of states, and the resulting increase of . The obmented in alkali metal–doped bulk samples servation of gate-induced hole doping of C resulting in a of 52 K suggests that significantly higher ’s could be anticipated in suitably “expanded” C crystals. Indeed, here is reto 117 K with such methods. ported a raise in Organic FETs (OFETs) are of great interest for future electroritic applications due to their flexibility. Up to now, a lot of fabrication processes or device configurations of OFETs have been reported [23]. Most of them were based on thin film technique such as vapor deposition. In general, the thin films for example, amorphous, poly crystalline, polymeric, and so on, consist of grains, and therefore, charge carriers behave as hopping conduction. This decreases the field-effect mobility of devices. Thus, annealing the organic thin films or surface preparations of substrate grows grains, and consequently these devices have few boundaries of grains between source and drain electrodes. A quite simple and easy fabrication technique of OFETs with single crystals was proposed, and it demonstrated that the OFETs exhibit high mobility and good device characteristics (Fig. 2). The presented technique makes the fabrication of high-performance OFETs easy and serves as a potential way to make organic integrated circuits. Also, N-channel and ambipolar OFETs with a few tens of nanometer channel length were fabricated and characterized [24]. The scientists fabricate FETs from CNT with the precise electrical properties and any variable bandgap desired. In parallel studies of CNT, researchers have been working to improve the electrical characteristics of individual nanotube transistors [25]. The realization of a self-assembled single-walled (SW) CNT
Oct. 1, 2014 Re: I-PRO-29.05.07 [complaint]
Dear Dr. Hüttemann, The procedure for the treatment of scientific misconduct by DFG appears completely subjective, without any relation to the highest standards of integrity, accuracy and fairness. First of all, the said "publicly presented concepts" were known to the DFG auditorium only, which is confirmed by absence of any citation in further publications. That is why I can not be sure in the degree of similarity between the mentioned old presentation and the plagiarized paper. Moreover, if even this is true, my method was presented in Sept. 1999, as a submission titled 'A n SFET Based Transducer of Nerve Impulses (The Living Being-Machine Interface Scheme as an Intelligent System's Term)' to “Shaping the Future” project of EXPO 2000 HANNOVER (enclosed please find an evident PDF). Secondly, it does not clear, how my arguments could "haven't been specific enough" if I have answered your question completely and any your further enquiries were not submitted during more than a year? Nevertheless, I am still agree to satisfy your requirements in the necessary details.
A key transducing element which was defined in the poster as "SFET" had been presented in XIII IMEKO World Congress and described in the relevant
publication:
'A cryogenic induction magnetometer', Proceedings of XIII IMEKO World Congress (International Measurement Confederation), September 5-9, 1994, Torino, Italy, pp. 2377-2382.
Yours sincerely,
Rostyslav SKLYAR, Dr. (Eng)
Nov. 1, 2014 Misconduct in research by Professor Dr.-Ing. Steffen Strehle
Dear Präsident Ebeling, The main idea of the paper titled "Outside Looking In: Nanotube Transistor Intracellular Sensors" (Nano Lett. 2012, 12, 3329−3333, DOI 10.1021/nl301623p) which was compiled a t Harvard University (USA) with participation of Universität Ulm by Professor Dr.-Ing. Steffen Strehle had been stolen from my paper “A CNTFET-based nanowired induction two-way
transducers",
published
on-line
in
ISPN
Nanotechnology
at
www.isrn.com/journals/nanotechnology/2012/102783, and the references within up to ten years old (enclosed please find two pages respectively for an evident understanding). It means that an already described gate output bioelectronic transducer has been renamed into some "an active silicon nanotube transistor, ANTT probe", as a result my invention was appropriated in an extremely impudent manner. Also this is further development of their previous impertinent cheating which has been revealed at http://issuu.com/r_sklyar/docs/harvard_mit . Despite of neglecting by the Ombudsman at Ulm (Prof. Dr. Lisa Wiesmüller and Prof. em. Dr. Hanns-Georg Kilian) their obligations during about one year and a half, I am still insisting on consideration of this case according to the highest standards of integrity, accuracy and fairness.
CC: Other relevant scientific authorities/principals Yours sincerely,
Rostyslav SKLYAR, Dr. (Eng)
Prof. em. Dr. Hanns-Georg Kilian Abteilung Experimentelle Physik Ombudsmann
D-89069 Ulm, 12. Jul. 201 3 Albert-Einstein Allee II Tel. 0731 502 6072 Fax. 0731 502 3036 mail : hanns-georg.kilain@physik.uni-ulm.de
An Professor Dr. Karl Joachim Ebeling (Präsident der Universität Ulm)
Betreff: Von R. Sklyar eine ungerechtfertigt scharf formulierte Beschuldigung einer Urheber-Verletzung.von Herrn Jun. Prof. Dr. Steffen Strehle. Sehr geehrter Präsident, lieber Herr Ebeling, Da Herr R. Sklyar unstatthaft "universitäts-öffentlich" Herrn Jun. Prof. Dr.-lng. Steffen Strehle (Institut für Elektronische Bauelemente und Schaltungen) schriftlich schwerwiegendes wissenschaftliches Fehlverhalten vorwirft, finden wir es am besten, jeden der Empfänger über das Ergebnis unserer gründlichen Untersuchung zu unterrichten.
Die pflichtgemäße Überprüfung durch mich als Ombudsmann in Absprache mit Herrn Strehle liefert den sicheren Nachweis, dass die Anschuldigung von Herrn R. Sklyar nicht haltbar ist (siehe Einzelheiten im Anhang). Herrn Jun. Prof. Dr.-lng. Steffen Strehle wird daher von mir als Ombudsmann der Universität Ulm unwiderruflich bescheinigt, dass der Vorwurf des schwerwiegenden wissenschaftlichen Fehlverhaltens nicht zutrifft.
Mit besten Grüßen. Ihr