Bioelectronic Silicon Nanowire Devices Using Functional Membrane Proteins

Bioelectronic silicon nanowire devices using functional membrane proteins

Nipun Misraa,b,1, Julio A. Martineza,c,1, Shih-Chieh J. Huanga,d, Yinmin Wanga, Pieter Stroevec, Costas P. Grigoropoulosb, and Aleksandr Noya,2

aPhysical and Life Sciences Directorate, Lawrence Livermore National Laboratory, Livermore, CA 94550; bDepartment of Mechanical Engineering, University of California, Berkeley, CA 94720; cDepartment of Chemical Engineering, University of California, Davis, CA 95616; and dDepartment of Civil Engineering, University of California, Los Angeles, CA 90095

Edited by Charles M. Lieber, Harvard University, Cambridge, MA, and approved June 23, 2009 (received for review May 1, 2009) Modern means of communication rely on electric fields and cur- number of protein machines that perform a large number of rents to carry the flow of information. In contrast, biological critical recognition, transport, and signal transduction functions systems follow a different paradigm that uses ion gradients and in the cell (13). Despite some initial work (14–16), the possibil- currents, flows of small molecules, and membrane electric poten- ities of using lipid membranes in nanoelectronic devices remain tials. Living organisms use a sophisticated arsenal of membrane virtually untapped. We have incorporated lipid bilayer mem- receptors, channels, and pumps to control signal transduction to a branes into SiNW transistors by covering the NW with a con- degree that is unmatched by manmade devices. Electronic circuits tinuous lipid bilayer shell that forms a barrier between the NW that use such biological components could achieve drastically surface and solution species. We show that when this ‘‘shielded increased functionality; however, this approach requires nearly wire’’ structure incorporates transmembrane peptide pores it seamless integration of biological and manmade structures. We enables ionic to electronic signal transduction by using voltage- present a versatile hybrid platform for such integration that uses gated and chemically gated ion transport through the membrane shielded nanowires (NWs) that are coated with a continuous lipid pores. bilayer. We show that when shielded silicon NW transistors incor- porate transmembrane peptide pores gramicidin A and alamethicin Results and Discussion in the lipid bilayer they can achieve ionic to electronic signal We have built our bionanoelectronic devices by using a micro- transduction by using voltage-gated or chemically gated ion trans- fabricated SiNW transistor platform. In these devices, a SiNW is port through the membrane pores. connected to a pair of metallic source and drain electrodes (Fig. 1 A and B). We used semiconducting p-doped SiNWs with the bionanoelectronics ͉ ion channels ͉ silicon nanowires ͉ lipid bilayers ͉ diameters in the 20- to 40-nm range (Fig. 1C) synthesized by membrane transport catalytic chemical vapor deposition (CVD) procedures that have been well-described in the literature (17). High quality of the iological systems use a combination of ion gradients, flows starting NW material is important for achieving high perfor- Bof small molecules, membrane electric potentials, and even mance of the transistor devices, and transmission electron light to achieve an astonishingly effective control over signal microscopy (TEM) images (Fig. 1C) showed that the NWs were transduction that is still largely unmatched by manmade devices. crystalline with a thin layer of native oxide on the surface. We To gain this level of sophistication nature has evolved a vast then fabricated NW transistors by depositing SiNWs on the arsenal of highly specific receptors, active and passive ion surface by using a flow-alignment procedure and then connect- channels (1, 2), photo-activated proton pumps, and ion pumps ing them to the source and drain electrodes by using conven- (3). Utilization of these components in electronic circuits could tional photolithography. Good isolation of the electrodes is achieve seamless integration of biological and manmade struc- critical for successful operation of these devices in liquid; therefore we passivated the source and drain electrodes by tures (4) that would enable superior biosensing and diagnostics coating them with a conformal 80-nm-thick layer of Si N (see tools (5), advanced neuroprosthetics (6), and more efficient 3 4 Methods for details). The finished chips then were mounted in a computers (7). Previous attempts at integrating biological sys- fluid cell that featured polydimethylsiloxane (PDMS) micro- tems with microelectronics range from the early works on channels for delivery of test solutions and a reference gate capacitive stimulation of cells (8) to monitoring neuronal activity electrode inserted in the solution channel. As-fabricated SiNW with field-effect transistors (FETs) (9) to a recent example of transistors show Ohmic contacts, on–off ratios of Ϸ104, and using nanowire (NW) transistor arrays to follow neuronal signal transconductances of 100–500 nS (Fig. 1D), comparable to the propagation (6). Nanomaterials that have characteristic dimen- parameters reported for similar devices operating in liquids (18). sions comparable to the size of biological molecules open up the The key procedure for building our bionanoelectronic device possibility of such integration at an even more localized level. was the formation of the lipid bilayer on the surface of the SiNW. Some early examples include using carbon nanotubes as carriers The hydrophilic negatively charged native oxide present on the for transporting intracellular proteins and DNA (10) and using NW surface in solution makes NWs particularly attractive as a silicon NWs (SiNWs) as gene delivery vehicles for mammalian template for supporting lipid bilayer formation. We have re- cells (11). Other work has taken advantage of the superior electronic properties of NWs to create specific electronic de- tectors for a variety of biomolecules (5). Author contributions: N.M., J.A.M., and A.N. designed research; N.M. and J.A.M. performed An important step toward building bionanoelectronic inter- research; S.-C.J.H. and Y.W. contributed new reagents/analytic tools; N.M., J.A.M., P.S., faces would involve functional integration of nanomaterials with C.P.G., and A.N. analyzed data; and N.M., J.A.M., and A.N. wrote the paper. membrane proteins. Lipid membranes occupy a special place in The authors declare no conflict of interest. the hierarchy of the cellular structures as they represent impor- This article is a PNAS Direct Submission. tant structural and protective elements of the cell that form a 1N.M. and J.A.M. contributed equally to this work. stable, self-healing, and virtually impenetrable barrier to the ions 2To whom the correspondence should be addressed. E-mail: [email protected]. and small molecules (12). However, a lipid membrane is also a This article contains supporting information online at www.pnas.org/cgi/content/full/ nearly universal matrix that can house a virtually unlimited 0904850106/DCSupplemental.

13780–13784 ͉ PNAS ͉ August 18, 2009 ͉ vol. 106 ͉ no. 33 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0904850106 Downloaded by guest on September 28, 2021 CHEMISTRY

Fig. 1. Bionanoelectronic devices incorporating lipid-coated SiNWs. (A) Device schematics showing a NW connected to microfabricated source (S) and drain (D) electrodes. (Insets) The conﬁguration of the lipid bilayer and a pore channel placed in the bilayer membrane. (B) An SEM micrograph of the NW transistor showing a NW bridging the source and drain electrodes. (Inset) A photograph of the device chip covered with a PDMS ﬂow channel. (C) TEM micrograph of the as-synthesized

SiNW. (D) A typical IV characteristic of the SiNW transistor in ﬂuid. (E) Cyclic voltammetry curves measured for an uncoated SiNW device (black line) and a device coated BIOPHYSICS AND

with the lipid bilayer (red line). Fe(CN)6 solution (10 mM) was used as a redox agent. (F) Time traces of the normalized conductance of an uncoated SiNW transistor (black COMPUTATIONAL BIOLOGY line) and SiNW transistor coated with the lipid bilayer (red) as the pH of the solution in the ﬂuid cell was changed from 6 to 9.

ported that unilamellar vesicles fuse onto a SiNW surface current at a given gate voltage (21). Bare NW FETs showed a producing a conformal lipid bilayer coating (19). Scanning pronounced increase in conductance when the pH of the fluidic confocal fluorescence microscopy images (see SI Appendix) environment around the NWs was changed from 6 to 9 (Fig. 1F), showed that 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) which corresponded to the average pH sensitivity of 50–100 lipid bilayers form a continuous coatings on the NWs, which for nS/pH in the pH range of 5 to 9. The observed response kinetics the surface-bound NWs is likely to be ‘‘omega’’-shaped (Fig. is not limited by the device speed; rather it reflects the kinetics 1A), and for the NWs suspended over a TEM grid hole it should of solution mixing in the fluid cell. Indeed, relative conductance resemble a core-shell NW-lipid structure. Significantly, in-plane traces measured in a particular experiment typically collapse on lipid molecule diffusion coefficients measured by fluorescence a single curve shape when normalized to the same initial and recovery after photobleaching (FRAP), Ϸ8 ␮m2/s on suspended final levels, which strongly argues that our kinetics are limited by wires and Ϸ5 ␮m2/s on substrate-bound NWs, were comparable the reagent delivery. to the values reported for the supported lipid bilayers on flat Note that protons are much smaller and much more diffusive 4Ϫ glass substrates (20). These results indicate that the lipid bilayers ionic species than most redox species, including Fe(CN)6 ; on SiNWs are highly fluid, which is a vital property that enables hence they present a much more stringent test to the shielding incorporation of biologically active structures into the mem- capacity of the lipid bilayers. Still, lipid bilayer formation on the brane. The second key property of the lipid bilayer is its ability SiNW surface led to an Ϸ10 times decrease in the FET response to shield the underlying NW surface from the solution species. to the pH changes in the fluid cell. These data indicate that the Indeed, cyclic voltammetry measurements using K4Fe(CN)6 as a DOPC bilayer membrane blocks proton transport between the redox probe showed that formation of the lipid bilayer on the fluid environment outside the lipid bilayer and the hydration NW surface reduced the limiting current by 85–95% relative to layer situated between the inner leaflet of the bilayer and SiNW the uncovered NW device (Fig. 1E). surface. To show that our devices can detect selective and specific The final element of our bioelectronic device platform is a transport of ions through the lipid membrane pore, we have membrane pore channel incorporated in the lipid bilayer. The exploited the sensitivity of the NW electrical response to the first device example that we report incorporates gramicidin A proton concentration in the solution. As the solution pH pores. Gramicidin A is a short helical polypeptide from Bacillus changes, charging of silanol groups at the silicon oxide layer on brevis, which can dimerize in the lipid membrane to form a the surface of p-type SiNWs leads to changes of the depletion transmembrane channel (Fig. 2A) that allows passage of small region in the SiNW channel that then affects the source-drain monovalent cations, while being impermeable to anions (22).

Misra et al. PNAS ͉ August 18, 2009 ͉ vol. 106 ͉ no. 33 ͉ 13781 Downloaded by guest on September 28, 2021 (22). Although some debate about the exact mechanism of the voltage-gated transport in ALM pores remains (25–27), research indicates that at positive applied transmembrane voltages on the insertion side (cis-side) ALM forms barrel-staved functional open pores big enough for small monovalent cations to diffuse through, whereas these pores do not form at zero bias or negative bias (25). Some evidence (28) indicates that at negative membrane potentials ALM helices do not span the entire transmembrane thickness, yet at the positive membrane potentials the helices tilt enough to penetrate the membrane completely and form a channel. We exploited this behavior in our devices by using the electric field applied by the device to open and close the ALM pores in the lipid bilayer. Indeed, at zero applied gate bias, ALM pores were ‘‘turned off’’ and the device response was nearly identical to the response of the NW coated with a pure lipid bilayer (Fig. 3B). However, when we applied a positive gate voltage of 150 mV to the device, we obtained a dramatically different response (Fig. 3C): the device showed a strong response to the pH change of up to 50% of the response observed for the bare NW, indicating that the ALM pores had been turned on. These results demonstrate that our device platform is not only capable of using biological components as functional parts of the device, but it also can control the functionality of the biological molecules, Fig. 2. Ligand-gated operation of devices incorporating gramicidin A pores. bringing the functional integration of the biological and nano- (A and B) Schematic showing proton transport in the bilayer incorporating a electronic components on a new level. gramicidin A pore in the absence (A) and presence (B)ofCa2ϩ ions. (C) Time It is tempting to assign the observed levels of signal change traces of normalized conductance of the SiNW device recorded as the solution to the differences in shielding of the NW surface by the lipid was changed from pH 5 to 7 for an uncoated NW device (red trace), a device bilayer with open ALM pores compared with the uncoated coated with lipid bilayer incorporating gramicidin A pores (blue trace), and a NW. In reality, the situation is likely more complicated. device coated with the lipid bilayer incorporating gramicidin A pores in Ionization of the silanol groups on the surface of the NW presence of Ca2ϩ ions (black trace). Dashed vertical line indicates the time when the pH of the ﬂuid cell input stream was switched from the lower to the produces immobile charges that interact with the mobile higher value. charges present on both sides of the lipid membrane and establish an electrical potential on the membrane in accor- dance with the Donnan membrane equilibrium (29). This The ionic conductance of the gramicidin channel can also be potential also influences the ionization equilibrium of the NW selectively blocked by divalent ions (22) such as Ca2ϩ that can surface, causing the effective pH on the inner side of the bind near the mouth of the pore (Fig. 2 A and B). Recent bilayer to be lower than on the outer side. This situation is experiments demonstrated that ionic conductance of gramicidin routinely observed in biological systems where Donnan equi- A channels could be used to gate macroscopic electrochemical librium causes the pH inside negatively charged lipid vesicles transistors (23). Similarly, incorporation of gramicidin A channel to be lower than pH of the outside solution (30). into the lipid bilayer NW FET device leads to a dramatic We can check the consistency of this interpretation by using recovery of the pH response of the NW FET device (Fig. 2C), it to estimate the potential on the membrane and compare it with indicating that functional ion channels were formed in the lipid the measured response of the NW transistor. The Donnan bilayer coating the NW. These data also confirm the long-range potential on the membrane is given by the Nernst equation: lateral fluidity of our lipid bilayer membranes, which is a RT ͓Cl͔ prerequisite for the successful transleaflet dimerization of gram- ⌬␾ ϭ i ln ͓ ͔ , [1] icidin A. Moreover, addition of 1 mM Ca2ϩ to the solution F Cl o dramatically alters the device behavior: the magnitude of the Ϸ where F is a Faraday constant, and [Cl]i(o) denotes the concen- response to pH change drops by 60% (Fig. 2C). These results tration of mobile chloride anions on the inner (outer) surface of are consistent with reports that show a 20–50% decrease in the membrane respectively, and [Cl] is given by: conductance of gramicidin A channels in the presence of Ca2ϩ i ions (24). Our measurements demonstrate that our bionano- ͓ ͔ 2 M 4C0 electronic device platform can successfully convert chemical ͓Cl͔ ϭ ⅐ ͩͱ1 ϩ Ϫ 1ͪ, [2] i 2 ͓M͔2 signals (pH change) into an electrical signal and that device efficiency can be regulated by using the same ligand-gating where [M] is the effective concentration of the immobile anions mechanism used by the biological systems. on the inner side of the membrane, and C0 is the concentration Another exciting possibility is to use the intrinsic electronic of the background electrolyte on the outside of the membrane functionality of the device itself to control ion transport through (see SI Appendix for the full derivation of Eq. 2). an ion channel in the lipid membrane surrounding the NW. We We can estimate the concentration of immobile ions, [M], by demonstrated this gating mechanism by using devices that using the calibration data on the NW pH response (31). incorporated self-inserting voltage-gated alamethicin (ALM) Conductance change values measured for proton transport pores. ALM is a peptide antibiotic from the fungus Trichoderma through ALM pores (Fig. 3B) correspond to the NW surface viride that is often used to mimic nerve cell action potential charge density increase of 0.2 electron/nm2. If we assume that across artificial membranes. ALM forms ion channels in lipid average thickness of the water layer between the NW surface bilayer membranes (Fig. 3A) by spontaneous insertion and and the lipid bilayer (32) is Ϸ1 nm, then this value corresponds to aggregation of 4–6 individual ALM helices into a helix bundle an effective concentration of immobile ions of 350 mM. For the

13782 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0904850106 Misra et al. Downloaded by guest on September 28, 2021 functionalities. This biomimetic device platform could thus enable new applications in biosensing, bioelectronics, neuroscience, and medicine. Methods NW Synthesis. Crystalline SiNWs were grown using the vapor liquid solid synthesis with gold colloidal particles as catalysts. Gold nanoparticles (20–30- nm; Ted Pella, Inc.) were dispersed onto a silicon wafer with 250-nm-thick oxide layer. The oxide layer was incubated in 0.1% aqueous solution of Poly-L-lysine (Ted Pella, Inc.) to promote adhesion of gold nanoparticles to the surface. After deposition of the gold nanoparticles, the substrate was rinsed with deionized (DI) water, dried with nitrogen, and cleaned in oxygen plasma to get clean catalyst surfaces. The NWs were grown at temperatures of 420–460 °C at 100 Torr. Boron-doped SiNWs were grown by ﬂowing 31 standard cubic cm per min (sccm) of 10% silane in helium (Voltaix) and 3 sccm of 100 ppm diborane in helium (Voltaix) for 30 min.

Device Fabrication and Characterization. After NW synthesis, the grown NWs were dispersed in ethanol by sonication for 5 s. The wires were then deposited onto silicon wafers with a 250-nm-thick dry-oxide layer grown at 1,200 °C. Poly-L-lysine functionalization was used to promote adhesion of the NWs to the substrate. The NW solution was flown onto the functionalized substrate in PDMS microfluidic channels on the desired areas of the substrate. After an oxygen plasma cleaning step, the deposited wires were annealed at 200 °C for 10 min to enhance adhesion. Metal contact regions were defined by photolithography, followed by e-beam deposition of Ti (10 nm)/Pt (70 nm) or Ni (80 nm). The contact regions were cleaned with oxygen plasma (100 sccm, 30 W), and the native oxide was etched away in these regions with a 5:1 buffered oxide etch for 10 s. An 80-nm conformal layer of stoichiometric silicon nitride was deposited onto the metal contacts by plasma enhanced CVD at 100 °C. CHEMISTRY After liftoff, the devices were further annealed in forming gas at 450 °C for 3 min to ensure good SiNW/metal contacts. The transistor performance in solution was tested in a DI water environment, with the solution gate voltage swept between Ϫ0.5 and 0.5 V by a leakage-free 3 M KCl Ag/AgCl reference microelectrode (Warner Instruments). All dc device measurements were done with a Keithley 2602 digital sourcemeter, and ac measurements were performed with a Stanford Research SR8650 lock-in amplifier at frequencies of 19 or 23 Hz. BIOPHYSICS AND

Lipid Bilayer Formation and Characterization. Unilamellar vesicles of DOPC lipid COMPUTATIONAL BIOLOGY were prepared by sonication of a solution of 2 mg/mL DOPC (Avanti Polar Lipids) in buffer. Vesicle fusion onto SiNWs was allowed for 24 h. The thickness of resulting bilayers was measured to be 4.4 nm on flat oxide surfaces by Fig. 3. Voltage-gated operation of the devices incorporating ALM pores. (A) surface plasmon resonance (SPR). Home-built systems were used to charac- Schematics showing the mechanism of voltage-gated proton transport in terize the lipid bilayers by fluorescence recovery after photobleaching, cyclic self-assembled ALM pores in the lipid bilayer. (B) Time traces of normalized voltammetry, and SPR. conductance of the SiNW device held at gate bias of 0V recorded as the solution was changed from pH 6 to 9 for the uncoated nanowire (blue trace), Ion Channel Incorporation in Lipid Bilayers. Gramicidin A (2 mg/mL; Biochemika; coated nanowire (black trace), and the coated NW device incorporating ALM Ͼ90% HPLC) in 200-proof ethanol was mixed with DOPC in chloroform followed pores. (C) Time traces of a similar experiment recorded at gate bias of 0.15 V. by solvent evaporation with N2 and buffer hydration. Unilamellar vesicles of Vertical dashed lines indicate the time when the pH of the fluid cell input DOPC/gramicidin mol ratio of 100:1 were formed by sonication. These vesicles stream was switched from the lower to the higher value. were then fused onto SiNW FET devices. ALM (Sigma; Ͼ90% HPLC) was incorporated into bilayers by spontaneous insertion by exposing pristine NW-bilayer structures to a solution of 5 mg/mL ALM for 30 min. 100-mM background electrolyte concentration used in our experiments, Eqs. 1 and 2 estimate the membrane potential as Ϫ34 mV. Electrical Measurements. Rapid exchange of fluids around the NW was If we use the IV characteristic of the NW transistor (Fig. 1D) achieved by bonding PDMS microfluidic channels on top of the device chip and to convert the measured conductance shifts into the effective by using suction from suitable fluid reservoirs by a syringe pump. Different pH solutions were prepared in 100 mM KCl and 5 mM PBS by the addition of NaOH gate voltage shifts, then we can estimate that the presence of the or HCL to adjust the pH. All pH values were measured by a calibrated benchtop membrane with ALM pores changes the effective gate voltage pH meter. All reagents were purchased from Sigma–Aldrich with the highest shift by Ϫ33 mV, the value that corresponds extremely well with purity available. our estimate of the Donnan membrane potential. Device architecture that comprises a NW device coated with Other Information. For confocal fluorescence microscopy images and FRAP a lipid bilayer incorporating functional membrane proteins data for suspended and surface bound SiNWs, SPR studies of lipid bilayer could serve as a versatile platform for building bionano formation, and derivation of Eqs. 1 and 2 see SI Appendix. interfaces that could enable direct conversion of biological ACKNOWLEDGMENTS: A.N. acknowledges support from Basic Energy Services signals into electronic impulses. Although we have used SiNW Biomolecular Materials Program and University of California-Lawrence Liver- devices and proton transport as a proof-of-concept example of more National Laboratory Research Program, and use of the facilities at the such a platform, it is possible to use other nanomaterials Molecular Foundry at Lawrence Berkeley National Laboratory. J.A.M. and and transport of other species to create different ion-sensitive S.-C.J.H. acknowledge support from the Lawrence Livermore National Labo- ratory Lawrence Scholar Program. Parts of this work were performed under designs. The lipid bilayers provide a matrix for a virtually unlimited the auspices of the U.S. Department of Energy by Lawrence Livermore Na- number of transmembrane proteins that can provide different tional Laboratory under Contract DE-AC52-07NA27344.

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